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Gene delivery is a complex process with several challenges when attempting to incorporate genetic material efficiently and safely into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal escape to ensure that the genetic material can exert its effect but also minimizing the toxicity of the delivery system, which is vital for safe gene delivery. Of importance, if gene delivery systems are intended for biomedical applications or clinical use, they must be scalable and easy and affordable to manufacture to meet the demand. Here, we show an efficient gene delivery method using a combination of carbon dots coated by PEI through electrostatic binding to easily generate cationic carbon dots. We show a biofunctional approach to generate optimal cationic carbon dots (CCDs) that can be scaled up to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency four times over the standard of PEI polyplexes. Generated CCDs enabled the challenging transfection protocol to produce retroviral vectors via cell cotransfection of three different plasmids into packing cells, showing not only high efficiency but also functionality of the gene delivery, tested as the capacity to produce infective retroviral particles.
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ABSTACTThe medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pKa. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.
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Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.
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Cadeias Pesadas de Miosina , Transfecção , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Humanos , Transfecção/métodos , Linhagem Celular , Animais , Camundongos , Miosinas CardíacasRESUMO
PURPOSE: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. METHODS: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) were determined, and selected powders were used for further characterization. RESULTS: Of the nine dry powders prepared, their MMAD values were about 1-2 µm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before TFF. Powders prepared with Tris-EDTA buffer or cosolvents (i.e., 1,4-dioxane or tert-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without Tris-EDTA were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix structure, with a moisture content of < 2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed that the activity of the pDNA did not change after it was subjected to TFF. CONCLUSION: It is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.
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DNA , Água , Pós/química , Administração por Inalação , Congelamento , Ácido Edético , Aerossóis/química , DNA/genética , Plasmídeos , Água/química , Tamanho da Partícula , Inaladores de Pó Seco/métodosRESUMO
BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a gastric malignancy with little relation to Helicobacter pylori. Clinical characteristics of GA-FG have been established, but molecular mechanisms leading to tumorigenesis have not yet been elucidated. METHODS: We subjected three GA-FG tumors-normal mucosa pairs to microarray analysis. Network analysis was performed for the top 30 up-regulated gene transcripts, followed by immunohistochemical staining to confirm the gene expression analysis results. AGS and NUGC4 cells were transfected with the gene-encoding NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) to evaluate transcriptional changes in its target genes. RESULTS: Comprehensive gene expression analysis identified 1410 up-regulated and 1395 down-regulated gene probes with ≥ two-fold difference in expression. Among the top 30 up-regulated genes in GA-FG, we identified transcription factor NKX2-1/TTF-1, a master regulator of lung/thyroid differentiation, together with surfactant protein B (SFTPB), SFTPC, and secretoglobin family 3A member 2(SCGB3A2), which are regulated by NKX2-1/TTF-1. Immunohistochemical analysis of 16 GA-FG specimens demonstrated significantly higher NKX2-1/TTF-1 and SFTPB levels, as compared to that in adjacent normal mucosa (P < 0.05), while SCGB3A2 levels did not differ (P = 0.341). Transduction of NKX2-1/TTF-1 into AGS and NUGC4 cells induced transactivation of SFTPB and SFTPC, indicating that NKX2-1/TTF-1 can function as normally in gastric cells as it can in the lung cells. CONCLUSIONS: Our first transcriptome analysis of GA-FG indicates significant expression of NKX2-1/TTF1 in GA-FG. Immunohistochemistry and cell biology show ectopic expression and normal transactivation ability of NKX2-1/TTF-1, suggesting that it plays an essential role in GA-FG development.
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Adenocarcinoma , Neoplasias Gástricas , Humanos , Fator Nuclear 1 de Tireoide/genética , Genes Homeobox , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Perfilação da Expressão GênicaRESUMO
Epithelial to mesenchymal transition (EMT) describes a process where epithelial tumor cells acquire mesenchymal characteristics. EMT often correlates with invasion and an increased cell migration potential by losing cellular polarity and cell-cell junctions. It is mainly induced by tumor-microenvironment factors, such as TGF-beta 1 and IL-6, which activate the increased expression of the EMT-transcription factor (TF) Slug. We previously reported the Slug/Krüppel-like factor 4 (KLF4) switch in EMT in HNSCC, and found, that in human papilloma virus (HPV)-negative HNSCC Slug gene expression was significant higher represented, than in HPV-positive HNSCC. The purpose of this study was to investigate the impact of KLF4 and Slug on the regulation of the cadherin switch and on the EMT phenotype. Gene expression of KLF4 positive correlated with E-cadherin in 71 head and neck squamous cell carcinoma (HNSCC) patient tissue samples, which we also confirmed by the investigation of the Cancer Genome Atlas database (TCGA). HPV-transcripts contributed to stabilization of KLF4 at protein level, and simultaneously upregulated E-cadherin. Furthermore, ectopic KLF4 overexpression was associated with epithelial gene expression by induction of E-cadherin, ß-catenin and 70-kDa heat shock protein (HSP-70). The presence of HSP-70 ensures the membranous localization of E-cadherin, therefore, the ability of cells to form cadherin/catenin complexes and cellular linkages. In conclusion, KLF4 is a major regulator of the epithelial cadherin-adhesion in HNSCC.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Caderinas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Infecções por Papillomavirus/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microambiente TumoralRESUMO
Chinese Hamster Ovary cells (CHO) have become the most common workhorse for the commercial production of therapeutic proteins, as well as for the production of recombinant proteins for biomedical research. The ability to grow at high density in suspension, the adaptability to serum free media, and the ease transfection and scale up, made CHO cell line highly productive and robust for large-scale production. Here, we present an optimized workflow used to successfully express and purify a number of human proteins with a yield up to 5 mg/L of culture. The entire protocol, from the synthetic gene design to the assessment of purified protein quality, can be completed in 2 weeks. The established cell culture platform has been efficiently adapted to rapidly produce the receptor-binding domain (RBD) in SARS-CoV-2 S protein, a protein required by many laboratories in 2020 to better understand the initial step of infection related to COVID-19 pandemic. An overall yield of 2 mg of high quality soluble RBD per liter of culture was obtained, a production 10-times cheaper than commercial preparations, this representing an intriguing strategy for future challenges.
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COVID-19 , Pandemias , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , SARS-CoV-2/genética , Proteínas Recombinantes , TransfecçãoRESUMO
OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.
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Actinas , Actinas/genética , Actinas/metabolismo , Citocalasina D/farmacologia , Transfecção , MembranasRESUMO
Gene therapy is extensively studied as a realistic and promising therapeutic approach for treating inherited and acquired diseases by repairing defective genes through introducing (transfection) the "healthy" genetic material in the diseased cells. To succeed, the proper DNA or RNA fragments need efficient vectors, and viruses are endowed with excellent transfection efficiency and have been extensively exploited. Due to several drawbacks related to their use, nonviral cationic materials, including lipidic, polymeric, and dendrimer vectors capable of electrostatically interacting with anionic phosphate groups of genetic material, represent appealing alternative options to viral carriers. Particularly, dendrimers are highly branched, nanosized synthetic polymers characterized by a globular structure, low polydispersity index, presence of internal cavities, and a large number of peripheral functional groups exploitable to bind cationic moieties. Dendrimers are successful in several biomedical applications and are currently extensively studied for nonviral gene delivery. Among dendrimers, those derived by 2,2-bis(hydroxymethyl)propanoic acid (b-HMPA), having, unlike PAMAMs, a neutral polyester-based scaffold, could be particularly good-looking due to their degradability in vivo. Here, an overview of gene therapy, its objectives and challenges, and the main cationic materials studied for transporting and delivering genetic materials have been reported. Subsequently, due to their high potential for application in vivo, we have focused on the biodegradable dendrimer scaffolds, telling the history of the birth and development of b-HMPA-derived dendrimers. Finally, thanks to a personal experience in the synthesis of b-HMPA-based dendrimers, our contribution to this field has been described. In particular, we have enriched this work by reporting about the b-HMPA-based derivatives peripherally functionalized with amino acids prepared by us in recent years, thus rendering this paper original and different from the existing reviews.
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Dendrímeros , Dendrímeros/química , Propionatos , Hempa , Transfecção , Técnicas de Transferência de Genes , Terapia GenéticaRESUMO
Agasicles hygrophila is a classical biological agent used to control alligator weed (Alternanthera philoxeroides). Previous research has indicated that the heat shock factor (HSF) is involved in regulating the transcriptional expression of Hsp70 in response to heat resistance in A. hygrophila. However, the regulatory mechanism by which AhHsf regulates the expression of AhHsp70 remains largely unknown. Here, we identified and cloned a 944 bp AhHsp70 promoter (AhHsp70p) region from A. hygrophila. Subsequent bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements and has a common TATA box approximately 30 bp upstream of the transcription start site, with transcription commencing at a purine base approximately 137 bp upstream of ATG. Promoter deletion analyses revealed that the sequence from -944 to -744 bp was the core regulatory region. A dual-luciferase reporter assay indicated that overexpressed AhHsf significantly enhanced the activity of AhHsp70p. Furthermore, qPCR showed that AhHsp70 expression increased with time in Spodoptera frugiperda (Sf9) cells, and AhHsf overexpression significantly upregulated AhHsp70 expression in vitro. Characterization of the upstream regulatory mechanisms demonstrated that AhHsf binds to upstream cis-acting elements in the promoter region of AhHsp70 from -944 to -744 bp to activate the AhHSF-AhHSP pathway at the transcriptional level to protect A. hygrophila from high temperature damage. Furthermore, we proposed a molecular model of AhHsf modulation of AhHsp70 transcription following heat shock in A. hygrophila. The findings of this study suggest that enhancing the heat tolerance of A. hygrophila by modulating the upstream pathways of the Hsp family can improve the biocontrol of A. philoxeroides.
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Acanthaceae , Amaranthaceae , Besouros , Termotolerância , Animais , Besouros/fisiologia , Termotolerância/genética , Fatores de Transcrição/genéticaRESUMO
Due to their stable fluorescence, biocompatibility, and amenability to functionalization, fluorescent nanodiamonds (FND) are promising materials for long term cell labeling and tracking. However, transporting them to the cytosol remains a major challenge, due to low internalization efficiencies and endosomal entrapment. Here, nanostraws in combination with low voltage electroporation pulses are used to achieve direct delivery of FND to the cytosol. The nanostraw delivery leads to efficient and rapid FND transport into cells compared to when incubating cells in a FND-containing medium. Moreover, whereas all internalized FND delivered by incubation end up in lysosomes, a significantly larger proportion of nanostraw-injected FND are in the cytosol, which opens up for using FND as cellular probes. Furthermore, in order to answer the long-standing question in the field of nano-biology regarding the state of the cell membrane on hollow nanostructures, live cell stimulated emission depletion (STED) microscopy is performed to image directly the state of the membrane on nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, which supports the hypothesis that many FND are delivered directly to the cytosol, avoiding endocytosis and lysosomal entrapment.
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Nanodiamantes , Nanoestruturas , Membrana Celular , Eletroporação , Endocitose , Corantes FluorescentesRESUMO
The rapid advances of genetic and genomic technology indicate promising therapeutic potential of genetic materials for regulating abnormal gene expressions causing diseases and disorders. However, targeted intracellular delivery of RNA therapeutics still remains a major challenge hindering the clinical translation. In this study, an elaborated plasmonic optoporation approach is proposed to efficiently and selectively transfect specific cells. The site-specific optoporation is obtained by tuning the spectral range of a supercontinuum pulsed picosecond laser in order for each individual cell binding gold nanostar with their unique resonance peak to magnify the local field strength in the near-infrared region and facilitate a selective delivery of small interfering RNA, messenger RNA, and Cas9-ribonucleoprotein into human retinal pigment epithelial cells. Numerical simulations indicate that optoporation is not due to a plasma-mediated process but rather due to a highly localized temperature rise both in time (few nanoseconds) and space (few nanometers). Taking advantage of the numerical simulation and fine-tuning of the optical strategy, the perforated lipid bilayer of targeted cells undergoes a membrane recovery process, important to retain their viability. The results signify the prospects of antibody functionalized nanostar-mediated optoporation as a simple and realistic gene delivery approach for future clinical practices.
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Ouro , RNA , Anticorpos , Técnicas de Transferência de Genes , Humanos , LasersRESUMO
Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called "Design of Transfections" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.
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Expressão Gênica , Células-Tronco Neurais/metabolismo , Plasmídeos/genética , Transfecção , Animais , Linhagem Celular Transformada , CamundongosRESUMO
Giardiavirus is the only virus that infects Giardia duodenalis, a highly prevalent parasite worldwide, especially in low-income and developing countries. This virus belongs to the Totiviridae family, being a relative of other viruses that infect fungi and protozoa. It has a simple structure with only two proteins encoded in its genome and it appears that it can leave the cell without lysis. All these characteristics make it an interesting study model; however, its research has unfortunately made little progress in recent years. Thus, in this review, we summarize the currently available data on Giardiavirus, from their structure, genome and main proteins, to the uses that have been given to them and the possible health applications for the future.
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Giardia lamblia/virologia , Giardiavirus/fisiologia , Animais , HumanosRESUMO
Induced pluripotent stem cells (iPSCs) bear great potential for disease modeling, drug discovery, and regenerative medicine; however, the wide adoption of iPSC for clinically relevant applications has been hindered by the extremely low reprogramming efficiency. Here, we describe a high-efficiency cellular reprogramming strategy by puncturing cells with an array of diamond nanoneedles, which is applied to temporally disrupt the cell membrane in a reversible and minimally invasive format. This method enables high-efficiency cytoplasmic delivery of mini-intronic plasmid vectors to initiate the conversion of human fibroblast cells to either primed iPSCs or naiÌve iPSCs. The nanopuncturing operation is directly performed on cells in adherent culture without any cell lift-off and is completed within just 5 min. The treated cells are then cultured in feeder-free medium to achieve a reprogramming efficiency of 1.17 ± 0.28%, which is more than 2 orders of magnitude higher than the typical results from common methods involving plasmid delivery.
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Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Fibroblastos , Vetores Genéticos , Humanos , PlasmídeosRESUMO
Biological therapies, such as recombinant proteins, are nowadays amongst the most promising approaches towards precision medicine. One of the most innovative methodologies currently available aimed at improving the production yield of recombinant proteins with minimization of costs relies on the combination of in silico studies to predict and deepen the understanding of the modified proteins with an experimental approach. The work described herein aims at the design and production of a biomimetic vector containing the single-chain variable domain fragment (scFv) of an anti-HER2 antibody fragment as a targeting motif fused with HIV gp41. Molecular modeling and docking studies were performed to develop the recombinant protein sequence. Subsequently, the DNA plasmid was produced and HEK-293T cells were transfected to evaluate the designed vector. The obtained results demonstrated that the plasmid construction is robust and can be expressed in the selected cell line. The multidisciplinary integrated in silico and experimental strategy adopted for the construction of a recombinant protein which can be used in HER2+-targeted therapy paves the way towards the production of other therapeutic proteins in a more cost-effective way.
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Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Simulação por Computador , Vetores Genéticos , Células HEK293 , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Humanos , Simulação de Acoplamento Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trastuzumab/genéticaRESUMO
OBJECTIVE: To investigate the effect of E74-like factor 5 (ELF5) overexpression on the growth and invasion ability of colorectal cancer cells and its effect on tumor formation in nude mice. METHODS: Human colorectal cancer SW480 and HT-29 cells were divided into 5 groups: the lentivirus (LV)- GFP group transfected with empty vector LV- GFP, the LV- ELF5 group transfected with recombinant LV- ELF5, the shRNA-NC group transfected with empty vector shRNA-NC, the shRNA- ELF5 group transfected with recombinant shRNA- ELF5, and the control group, not transfected with any vector. Seventy-two h after transfection, the cell supernatant containing lentivirus was collected. The mRNA expression level of ELF5 in each group was examined by real-time fluorescent quantitative PCR (RT-qPCR). The protein expression levels of ELF5, apoptosis-related cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9, and invasion-related E-cadherin and N-cadherin were checked with Western blot. CCK-8 was used to check cell viability. Colony formation experiment was done to evaluate colony formation rate. Flow cytometry was used to assess cell apoptosis. Transwell migration assay was used to examine cell invasion. TUNEL assay was used to examine the apoptosis of tissues cells. Immunohistochemistry test was done to determine the expression of E-cadherin and N-cadherin in tissues. 20 BALB/c nude mice were put into 4 groups (5 in each group): LV- GFP group, shRNA-NC group, LV- ELF5 group, and shRNA- ELF5 group. Recombinant lentiviral SW480 cell supernatants were subcutaneously injected into nude mice to construct nude mice tumorigenesis models and the volume changes of transplanted tumors were monitored. On the 30th day, transplanted tumor tissues from the nude mice were extracted and the tumor mass was measured. Western blot was done to measure the expression of ELF4 protein in the transplanted tumors. TUNEL staining was used to check cell apoptosis in the tissues, and the positive expression of N-cadherin in the transplanted tumor was measured by immunohistochemical tests. RESULTS: Compared with the control group, there was no statistically significant difference in the indicators of the two cell lines in the LV- GFP group and shRNA-NC group. The results of Western blot and RT-qPCR showed that the ELF5 protein and mRNA of the LV- ELF5 group of the two cell lines were up-regulated ( P<0.05, compared with those of the LV- GFP group), and the ELF5 protein and mRNA of the shRNA- ELF5 group were down-regulated ( P<0.05). The ELF5 overexpression system and interference system were successfully constructed. Compared with the LV- GFP group, data from the LV- ELF5 group showed that cell viability and colony formation rate ( P<0.05) were reduced, SW480 and HT-29 cell apoptosis was promoted, cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 protein expression was up-regulated ( P<0.05), cell invasion was inhibited, and the expression of E-cadherin protein was up-regulated while the expression of N-cadherin protein was down-regulated ( P<0.05). After ELF5 interference, the above-mentioned expression of cells demonstrated an opposite trend ( P<0.05, comparing shRNA- ELF5 group with shRNA-NC group). In vivo experimental results indicated that ELF5 overexpression reduced tumor volume and tumor mass ( P<0.05), promoted cell apoptosis in tissues ( P<0.05), and inhibited N-cadherin protein expression ( P<0.05). When ELF5 expression was inhibited, the above mentioned experimental results showed the opposite trend. CONCLUSION: In vivo and in vitro experiments showed that ELF5 overexpression could promote the apoptosis of colorectal cancer cells and inhibit the growth and invasion of colorectal cancer cells.
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Neoplasias do Colo , Fator V , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/genética , Proteínas de Ligação a DNA , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Transcrição , TransfecçãoRESUMO
Polymeric nanoparticles have aroused an increasing interest in the last decades as novel advanced delivery systems to improve the treatment of many diseases. Hard work has been performed worldwide designing and developing polymeric nanoparticles using different building blocks, which target specific cell types, trying to avoid bioaccumulation and degradation pathways. The main handicap of the design is to understand the final fate and the journey that the nanoparticle will follow, which is intimately ligated with the chemical and physical properties of the nanoparticles themselves and specific factors of the targeted cells. Although the huge number of published scientific articles regarding polymeric nanoparticles for biomedical applications, their use in clinics is still limited. This fact could be explained by the limited data reporting the interaction of the huge diversity of polymeric nanoparticles with cells. This knowledge is essential to understand nanoparticle uptake and trafficking inside cells to the subcellular target structure.In this chapter, we aim to contribute to this field of knowledge by: (1) summarizing the polymeric nanoparticles properties and cellular factors that influence nanoparticle endocytosis and (2) reviewing the endocytic pathways classified as a function of nanoparticle size and as a function of the receptor playing a role. The revision of previously reported endocytic pathways for particular polymeric nanoparticles could facilitate scientist involved in this field to easily delineate efficient delivery systems based on polymeric nanoparticles.
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Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Nanopartículas/metabolismo , Polímeros/farmacocinética , Transporte Biológico , Endocitose , Humanos , Nanopartículas/administração & dosagem , Polímeros/administração & dosagemRESUMO
Hunter syndrome (mucopolysaccharidosis II; MPS II) is caused by a defect of the iduronate-2-sulfatase (IDS) gene. Few studies have reported integrated mutation data of Taiwanese MPS II phenotypes. In this study, we summarized genotype and phenotype correlations of confirmed MPS II patients and asymptomatic MPS II infants in Taiwan. Regular polymerase chain reaction and DNA sequencing were used to identify genetic abnormalities of 191 cases, including 51 unrelated patients with confirmed MPS II and 140 asymptomatic infants. IDS activity was analyzed in individual novel IDS variants using in vitro expression studies. Nineteen novel mutations were identified, in which the percentages of IDS activity of the novel missense mutations c.137A>C, c.311A>T, c.454A>C, c.797C>G, c.817C>T, c.998C>T, c.1106C>G, c.1400C>T, c.1402C>T, and c.1403G>A were significantly decreased (p < 0.001), c.254C>T and c.1025A>G were moderately decreased (p < 0.01), and c.851C>T was slightly decreased (p < 0.05) comparing with normal enzyme activity. The activities of the other six missense mutations were reduced but were insignificant. The results of genomic studies and their phenotypes were highly correlated. A greater understanding of the positive correlations may help to prevent the irreversible manifestations of Hunter syndrome, particularly in infants suspected of having asymptomatic MPS II. In addition, urinary glycosaminoglycan assay is important to diagnose Hunter syndrome since gene mutations are not definitive (could be non-pathogenic).
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Glicoproteínas/metabolismo , Mucopolissacaridose II , Mutação de Sentido Incorreto , Povo Asiático , Feminino , Glicoproteínas/genética , Humanos , Lactente , Masculino , Mucopolissacaridose II/enzimologia , Mucopolissacaridose II/genética , Mucopolissacaridose II/urina , Análise de Sequência de DNA , TaiwanRESUMO
Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier for DNA transfer thanks to its strong interaction with DNA molecules and its ability to be naturally internalized into mammalian cells through both non-endocytic and endocytic pathways. Sepiolite, due to its ability to bind various biomolecules, could be a good candidate for use as a nanocarrier for the simultaneous vectorization of diverse biological molecules. In this paper, we review our recent work, issued from a starting collaboration with Prof. Ruiz-Hitzky, that includes diverse aspects on the characterization and main features of sepiolite/DNA nanohybrids, and we present an outlook for the further development of sepiolite for DNA transfer.