Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30.528
Filtrar
1.
J Phys Chem Lett ; 12(1): 717-723, 2021 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-33400538

RESUMEN

Radiobiological damage is principally triggered by an initial cation and a secondary electron (SE). We address the fundamental questions: What lesions are first produced in DNA by this cation or nonionizing SE? What are their relative contributions to isolated and potentially lethal cluster lesions? Five monolayer films of dry plasmid DNA deposited on graphite or tantalum substrates are bombarded by 0.1-100 eV electrons in a vacuum. From measurements of the current transmitted through the films, 3.5 and 4.5 cations per incident 60 and 100 eV electrons, respectively, are estimated to be produced and stabilized within DNA. Damage analysis at 6, 10, 20, 30, 60, and 100 eV indicates that essentially all lesions, but preferentially cluster damages, are produced by non-ionizing or weakly ionizing electrons of energies below 12 eV. Most of these lesions are induced within femtosecond times, via transient anions and electron transfer within DNA, with little contributions from the numerous cations.


Asunto(s)
Daño del ADN , Electrones , Radiobiología , Cinética , Plásmidos/genética
2.
Int J Nanomedicine ; 16: 345-357, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33488076

RESUMEN

Background: Our previous study found that deletion of Sorting nexin 10 (SNX10) can protect against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). This inspired us that modulation of SNX10 expression in colonic epithelial cells might represent a promising therapeutic strategy for inflammatory bowel disease (IBD). Methods: Effective delivery of siRNA/shRNA to silence genes is a highly sought-after means in the treatment of multiple diseases. Here, we encapsulated SNX10-shRNA plasmids (SRP) with polylactide-polyglycolide (PLGA) to make oral nanoparticles (NPs), and then applied them to acute and chronic IBD mice model, respectively. The characteristics of the nanoparticles were assayed and the effects of SRP-NPs on mouse IBD were evaluated. Results: High-efficiency SNX10-shRNA plasmids were successfully constructed and coated with PLGA to obtain nanoparticles, with a particle size of 275.2 ± 11.4mm, uniform PDI distribution, entrapment efficiency of 87.6 ± 2.5%, and drug loading of 13.11 ± 1.38%, displayed dominant efficiency of SNX10 RNA interference in the colon. In both acute and chronic IBD models, SRP-NPs could effectively reduce the loss of mice body weight, relieve the intestinal mucosal damage and inflammatory infiltration, inhibit the expression of inflammatory cytokines IL-1ß, IL-23, TNF-α, and down-regulate the expression of toll-like receptors (TLRs) 2 and 4. Conclusion: Oral nanoparticles of SNX10-shRNA plasmid displayed dominant efficiency of SNX10 RNA interference in the colon and ameliorate mouse colitis via TLR signaling pathway. SNX10 is a new target for IBD treatment and nanoparticles of SNX10-shRNA plasmid might be a promising treatment option for IBD.


Asunto(s)
Colitis/terapia , Portadores de Fármacos/química , Terapia Genética/métodos , Nanopartículas/química , Plásmidos/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Administración Oral , Animales , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Ratones , Interferencia de ARN , ARN Interferente Pequeño/química , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/metabolismo
3.
Int J Nanomedicine ; 16: 421-432, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33488078

RESUMEN

Purpose: To synthesize echogenic chitosan/perfluorohexane nanodroplets (CNDs) for DKK-2 gene delivering in a spatiotemporally controlled manner in vitro. Methods: The characteristics, contrast-enhanced ultrasound imaging, DNA binding and DNase protection capacity, DKK-2 gene transfection and effects on LNCaP cells of these CNDs were investigated. Results: The obtained CNDs showed positive surface charges and could attract the genetic cargo with negative surface charges to form nanocomplexes. Agarose gel electrophoresis confirmed binding of the CNDs and pDNA. DKK-2 pDNA-loaded CNDs, in combination with ultrasound, ruptured and released DKK-2 pDNA, entering LNCaP cells through nano-scale pores in the cell membrane, which further reduced the proliferation of LNCaP cells. Conclusion: These stable and safe CNDs may be a promising choice to achieve efficient ultrasound-mediated gene delivery to specific tissues in a spatiotemporally controlled manner.


Asunto(s)
Quitosano/química , Portadores de Fármacos/química , Péptidos y Proteínas de Señalización Intercelular/genética , Nanopartículas/química , Neoplasias de la Próstata/patología , Ondas Ultrasónicas , Fluorocarburos/química , Humanos , Masculino , Plásmidos/genética , Transfección
4.
Nucleic Acids Res ; 49(2): 832-846, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406256

RESUMEN

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDCSGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriVSGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter PS004. This replicon transiently maintains SGI1 as a 4-8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.


Asunto(s)
Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Conjugación Genética/genética , Farmacorresistencia Bacteriana Múltiple/genética , Secuencias Repetitivas Esparcidas/genética , Salmonella typhimurium/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros , Integrasas/metabolismo , Operón/genética , Filogenia , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Recombinasas/metabolismo , Replicón/genética , Alineación de Secuencia , Transactivadores/genética , Transactivadores/metabolismo
5.
Int J Nanomedicine ; 16: 185-199, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33447034

RESUMEN

Background: Therapy for glioblastoma (GBM) has always been very challenging, not only because of the presence of the blood-brain barrier (BBB) but also due to susceptibility to drug resistance. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) has revolutionized gene editing technology and is capable of treating a variety of genetic diseases, including human tumors, but there is a lack of safe and effective targeting delivery systems in vivo, especially in the central nervous system (CNS). Methods: Lipid-polymer hybrid nanoparticles (LPHNs-cRGD) were constructed for efficient and targeting delivery of CRISPR/Cas9 plasmids targeting O6-methylguanine-DNA methyltransferase (MGMT), a drug-resistance gene to temozolomide (TMZ). Focused ultrasound (FUS)-microbubbles (MBs) were used to non-invasively and locally open the BBB to further facilitate gene delivery into glioblastoma in vivo. The gene editing efficiency and drug sensitivity changes were evaluated both in vitro and in vivo. Results: The gene-loaded LPHNs-cRGD were successfully synthesized and could protect pCas9/MGMT from enzyme degradation. LPHNs-cRGD could target GBM cells and mediate the transfection of pCas9/MGMT to downregulate the expression of MGMT, resulting in an increased sensitivity of GBM cells to TMZ. MBs-LPHNs-cRGD complexes could safely and locally increase the permeability of the BBB with FUS irradiation in vivo and facilitated the accumulation of nanoparticles at the tumor region in orthotopic tumor-bearing mice. Furthermore, the FUS-assisted MBs-LPHNspCas9/MGMT-cRGD enhanced the therapeutic effects of TMZ in glioblastoma, inhibited tumor growth, and prolonged survival of tumor-bearing mice, with a high level of biosafety. Conclusion: In this work, we constructed LPHNs-cRGD for targeting delivery of the CRISPR/Cas9 system, in combination with FUS-MBs to open the BBB. The MBs-LPHNs-cRGD delivery system could be a potential alternative for efficient targeting gene delivery for the treatment of glioblastoma.


Asunto(s)
Neoplasias Encefálicas/terapia , Resistencia a Antineoplásicos , Terapia Genética , Glioblastoma/terapia , Ultrasonido Enfocado de Alta Intensidad de Ablación , Lípidos/química , Nanopartículas/química , Polímeros/química , Animales , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Desoxirribonucleasa I/metabolismo , Glioblastoma/tratamiento farmacológico , Humanos , Ratones Endogámicos NOD , Ratones SCID , Microburbujas , Nanopartículas/ultraestructura , Péptidos Cíclicos/química , Plásmidos/genética , Temozolomida/farmacología , Temozolomida/uso terapéutico , Distribución Tisular , Transfección
6.
Int J Nanomedicine ; 16: 359-370, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33469290

RESUMEN

Purpose: Gold nanoparticles (AuNPs) are candidate radiosensitizers for medium-energy photon treatment, such as γ-ray radiation in high-dose-rate (HDR) brachytherapy. However, high AuNP concentrations are required for sufficient dose enhancement for clinical applications. Here, we investigated the effect of positively (+) charged AuNP radiosensitization of plasmid DNA damage induced by 192Ir γ-rays, and compared it with that of negatively (-) charged AuNPs. Methods: We observed DNA breaks and reactive oxygen species (ROS) generation in the presence of AuNPs at low concentrations. pBR322 plasmid DNA exposed to 64 ng/mL AuNPs was irradiated with 192Ir γ-rays via HDR brachytherapy. DNA breaks were detected by observing the changes in the form of the plasmid and quantified by agarose gel electrophoresis. The ROS generated by the AuNPs were measured with the fluorescent probe sensitive to ROS. The effects of positively (+) and negatively (-) charged AuNPs were compared to study the effect of surface charge on dose enhancement. Results: +AuNPs at lower concentrations promoted a comparable level of radiosensitization by producing both single-stranded breaks (SSBs) and double-stranded breaks (DSBs) than those used in cell assays and Monte Carlo simulation experiments. The dose enhancement factor (DEF) for +AuNPs was 1.3 ± 0.2 for SSBs and 1.5 ± 0.4 for DSBs. The ability of +AuNPs to augment plasmid DNA damage is due to enhanced ROS generation. While -AuNPs generated similar ROS levels, they did not cause significant DNA damage. Thus, dose enhancement using low concentrations of +AuNPs presumably occurred via DNA binding or increasing local +AuNP concentration around the DNA. Conclusion: +AuNPs at low concentrations displayed stronger radiosensitization compared to -AuNPs. Combining +AuNPs with 192Ir γ-rays in HDR brachytherapy is a candidate method for improving clinical outcomes. Future development of cancer cell-specific +AuNPs would allow their wider application for HDR brachytherapy.


Asunto(s)
Braquiterapia , Daño del ADN , Oro/farmacología , Nanopartículas del Metal/química , Plásmidos/genética , Fármacos Sensibilizantes a Radiaciones/farmacología , Dosificación Radioterapéutica , Simulación por Computador , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Humanos , Radioisótopos de Iridio/química , Nanopartículas del Metal/ultraestructura , Método de Montecarlo , Especies Reactivas de Oxígeno/metabolismo
7.
Microbiome ; 9(1): 3, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397505

RESUMEN

An article published in Microbiome in July 2018 uses incorrect definitions of integron integrase IntI1 and of class 1 integrons that affect the interpretation of the data.


Asunto(s)
Genoma Bacteriano , Integrones , Farmacorresistencia Microbiana , Genoma Bacteriano/genética , Integrones/genética , Filogenia , Plásmidos/genética
8.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 178-186, 2021 Jan 25.
Artículo en Chino | MEDLINE | ID: mdl-33501799

RESUMEN

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 107·667 TCID50/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 106·667 TCID50/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.


Asunto(s)
Virus del Moquillo Canino , Animales , Chlorocebus aethiops , Células Clonales , ADN Complementario , Virus del Moquillo Canino/genética , Plásmidos/genética , Células Vero
9.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 321-330, 2021 Jan 25.
Artículo en Chino | MEDLINE | ID: mdl-33501812

RESUMEN

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.


Asunto(s)
Escherichia coli , Vectores Genéticos , Clonación Molecular , Escherichia coli/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Operón Lac/genética , Plásmidos/genética , beta-Galactosidasa/genética
10.
Environ Pollut ; 270: 116296, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-33341549

RESUMEN

Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 µg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.


Asunto(s)
Transferencia de Gen Horizontal , Material Particulado , Animales , Antibacterianos , Hospitales , Plásmidos/genética , Prevalencia , Porcinos
11.
Int J Food Microbiol ; 337: 108956, 2021 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-33189985

RESUMEN

There has been an increase in the number of reports on Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) isolated from animals and humans. Recent studies using whole genome sequencing (WGS) have provided evidence on the likely contribution of a unique conjugative megaplasmid (pESI; ~280 kb) to the dissemination of this serovar worldwide. In the present study, twenty-two unrelated Salmonella strains [S. Infantis (n = 20) and Salmonella 6,7:r:- (n = 2)] and their plasmids were investigated using next generation sequencing technologies (MiSeq and MinION) to unravel the significant expansion of this bacteria in Turkey. Multi-locus sequence typing, plasmid replicons, resistance gene contents as well as phylogenetic relations between strains were determined. According to the WGS data, all S. Infantis possessed the relevant megaplasmid backbone genes and belonged to sequence type 32 (ST32) with the exception of a single novel ST7091. Tetracycline and trimethoprim/sulfamethoxazole resistance were found to be widespread in S. Infantis strains and the resistant strains exclusively carried the tetA, sul1, sul2 and dfrA14 genes. One S. Infantis isolate was also a carrier of the plasmid-mediated ampC via blaCMY-2, gene. Moreover, full genomes of four S. Infantis isolates were reconstructed based on hybrid assembly. All four strains contained large plasmids (240-290 kb) similar to previously published megaplasmid (pESI) and accompanied by several small plasmids. The megaplasmid backbone contained a toxin-antitoxin system, two virulence cassettes and segments associated with heavy metals resistance, while variable regions possessed several antibiotic resistance genes flanked by mobile elements. This study indicated that pESI-like megaplasmid is widely disseminated within the tested S. Infantis strains of chicken meat, warranting further genomic studies on clinical strains from humans and animals to uncover the overall emergence and spread of this serovar.


Asunto(s)
Genoma Bacteriano/genética , Plásmidos/genética , Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Salmonella/genética , Animales , Antibacterianos/farmacología , Pollos/microbiología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Microbiología de Alimentos , Filogenia , Plásmidos/efectos de los fármacos , Salmonella/efectos de los fármacos , Salmonella/aislamiento & purificación , Salmonella/patogenicidad , Salmonelosis Animal/epidemiología , Turquia/epidemiología , Virulencia/genética
12.
Gene ; 772: 145386, 2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33373662

RESUMEN

The emergence of community acquired infections increases the public health concern on K. pneumoniae and closely related bacteria among which antimicrobial resistance spreads. We report a multidrug-resistant K. pneumoniae isolate, B31, of a patient infected in the community and admitted to an intensive care unit in Northeast Brazil. Antimicrobial susceptibility and genome information were thoroughly investigated to characterize B31 in front of 172 sequenced strains of different countries. Assigned to the Sequence Type 15, which is globally spread, B31 presented extended spectrum beta-lactamase, tigecycline and ciprofloxacin resistance. Genome sequencing revealed most resistance genes being carried by plasmids with high dissemination potential. The absence of main virulence factors, like yersiniabactin and colibactin, apparently suggests a mild pathogenic strain which, on the contrary, persisted and caused severe infection in a previously healthy patient. The present study contributes to unveil the unclear genomic scenario of virulent and multidrug-resistant K. pneumoniae in Brazil.


Asunto(s)
Infecciones Comunitarias Adquiridas/microbiología , Farmacorresistencia Bacteriana Múltiple , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/clasificación , Secuenciación Completa del Genoma/métodos , Adulto , Ciprofloxacino/farmacología , Femenino , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Klebsiella pneumoniae/genética , Tipificación de Secuencias Multilocus , Plásmidos/genética , Tigeciclina/farmacología
13.
J Vis Exp ; (166)2020 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-33346188

RESUMEN

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).


Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Genes Reporteros , Luciferasas/metabolismo , Mamíferos/metabolismo , Plásmidos/genética , Animales , Secuencia de Bases , Línea Celular , ADN/metabolismo , Reparación del ADN , Vectores Genéticos/metabolismo , Luciferasas/genética , Oligonucleótidos/metabolismo , ARN Guia/genética , Reproducibilidad de los Resultados , Ovinos , Transformación Genética
14.
Int J Nanomedicine ; 15: 10305-10320, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33376323

RESUMEN

Purpose: The clinical management of patients with castration-resistant prostate cancer (CRPC) is difficult. However, novel treatment methods are gradually being introduced. Considering the adverse effects of traditional treatments, recent studies have investigated gene therapy as a method to combat CRPC; but, the application of long non-coding (lnc) RNA in gene therapy remains scarce, despite their promise. Therefore, it is imperative to develop a system that can efficiently deliver lncRNA for the treatment of CRPC. Here, we investigated the efficacy of a delivery system by introducing the plasmid-encoding tumor suppressor lncRNA MEG3 (pMEG3) in CRPC cells. Materials and Methods: An EpDT3 aptamer-linked poly(amidoamine) (PAMAM) dendrimer targeting EpCAM was used to deliver pMEG3 in CRPC cells. The PAMAM-PEG-EpDT3/pMEG3 nanoparticles (NPs) were tested using in vitro cellular assays including cellular uptake, entry, and CCK-8 measurement, and tumor growth inhibition, histological assessment, and safety evaluations in in vivo animal models. Results: The EpDT3 aptamer promoted endocytosis of PAMAM and PAMAM-PEG-EpDT3/pMEG3 NPs in CRPC cells. PAMAM-PEG-EpDT3/pMEG3 NPs exhibited a significant anti-CRPC effect, both in vivo and in vitro, when compared to that of unfunctionalized PAMAM-PEG/pMEG3 NPs. Conclusion: PAMAM-PEG-EpDT3/pMEG3 NPs can potentially improve gene therapy in CRPC cells.


Asunto(s)
Aptámeros de Nucleótidos/química , Dendrímeros/química , Terapia Genética/métodos , Plásmidos/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/terapia , ARN Largo no Codificante/genética , Animales , Línea Celular Tumoral , Portadores de Fármacos/química , Humanos , Masculino , Nanopartículas/química , Plásmidos/química , Polietilenglicoles/química
15.
Proc Natl Acad Sci U S A ; 117(52): 32919-32928, 2020 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-33318196

RESUMEN

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer-DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer-pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.


Asunto(s)
Acrilatos/química , Técnicas de Transferencia de Gen , Plásmidos/genética , Quinina/química , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Células K562
16.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2193-2205, 2020 Oct 25.
Artículo en Chino | MEDLINE | ID: mdl-33169583

RESUMEN

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.


Asunto(s)
Celulasa , Ingeniería Genética , Microbiología Industrial , Saccharomyces cerevisiae , Celulasa/genética , Plásmidos/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(10): 884-889, 2020 Oct.
Artículo en Chino | MEDLINE | ID: mdl-33148382

RESUMEN

Objective To analyze the physicochemical properties, structure and function of melanoma-associated antigen D4 (MAGE-D4) protein, and then construct the eukaryotic expression vector of MAGE-D4. Methods The physicochemical properties, structure and function of MAGE-D4 protein were analyzed by bioinformatics. Using MAGE-D4/pMAL-C2 prokaryotic recombinant plasmid as the template, PCR product digested by restriction enzyme was connected with pEGFP-C1 eukaryotic expression plasmid and transformed into E. coli. Ligation products were identified by antibiotic screening, enzyme digestion and sequencing. Then the recombinant plasmid was transfected into A549 lung cancer cells by liposome. Results MAGE-D4 protein was an unstable hydrophilic protein without transmembrane structure and signal peptide. Its secondary structure was mainly α-helix. MAGE-D4 contained multiple functional modification sites and was mainly located in the nucleus. SLLLVILGV might be a restricted T cell epitope of HLA-A*0201 derived from MAGE-D4. The first three proteins to potentially interact with MAGE-D4 were NSMCE4A, MLANA/MART-1 and BAGE5. DNA sequencing showed that the recombinant plasmid contained full-length coding sequence (CDS) of MAGE-D4 and it could be successfully transfected into A549 lung cancer cells. Conclusion MAGE-D4 protein is an unstable nuclear protein, which may play functions by interacting with a variety of melanoma-related proteins. The peptide derived from MAGE-D4 may have strong immunogenicity. The eukaryotic expression vector of MAGE-D4 has been successfully constructed.


Asunto(s)
Antígenos de Neoplasias/genética , Biología Computacional , Vectores Genéticos , Proteínas de Neoplasias/genética , Células A549 , Epítopos de Linfocito T , Escherichia coli , Eucariontes , Vectores Genéticos/genética , Humanos , Plásmidos/genética , Transfección
18.
Mem Inst Oswaldo Cruz ; 115: e200370, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33174903

RESUMEN

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.


Asunto(s)
Bacillus anthracis/aislamiento & purificación , ADN Bacteriano/genética , Plásmidos/análisis , Reacción en Cadena de la Polimerasa/métodos , Microbiología del Suelo , Esporas Bacterianas , Factores de Virulencia/genética , Antígenos Bacterianos , Bacillus anthracis/genética , Bacillus anthracis/patogenicidad , Toxinas Bacterianas , Brasil , ADN Bacteriano/análisis , Humanos , Plásmidos/genética , Análisis de Secuencia de ADN , Suelo , Virulencia
19.
Huan Jing Ke Xue ; 41(8): 3748-3757, 2020 Aug 08.
Artículo en Chino | MEDLINE | ID: mdl-33124350

RESUMEN

In order to explore the conjugation of genes encoding extended-spectrum ß-lactamase (ESBL), ESBL-expressing P. aeruginosa and E.coli strains isolated from the wastewater of major hospitals in Singapore were used as donors. gfp-tagged E.coli SCC1 strains resistant to chloramphenicol (CHL) were chosen as recipients. Using response surface analysis, we detected and analyzed the induction of conjugal transfer under single-exposure and co-exposure of tetracycline (TC), sulfamethoxazole (SMZ), and ceftazidime (CAZ) at sublethal concentrations. It was found that the ESBL plasmid could be conjugal transferred from P. aeruginosa and E.coli strains to the recipient E.coli SCC1 strains at an average frequency of 0.0015 and 0.0042, respectively, without stress from inducing antibiotics, thus showing a low fitness cost and higher conjugal frequency between E.coli strains under the exposure of sub-MIC antibiotics. A significant conjugation between E.coli strains occurred under the single-exposure or co-exposure of a TC concentration of <0.03 mg·L-1 and a CAZ concentration of <0.002 mg·L-1, as inhibited by a sub-MIC level of TC. The conjugation between P. aeruginosa and E.coli strains was stimulated under the exposure of TC and CAZ with concentrations 5-times larger than the MIC, while no significant induction was detected from the sub-MIC antibiotics.


Asunto(s)
Antibacterianos , Transferencia de Gen Horizontal , Antibacterianos/toxicidad , Ceftazidima , Escherichia coli/genética , Plásmidos/genética , beta-Lactamasas/genética
20.
Rev Soc Bras Med Trop ; 53: e20200397, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33111914

RESUMEN

INTRODUCTION: Antibiotic resistance in carbapenemase-producing Klebsiella pneumoniae is acquired and disseminated mainly by plasmids. Therefore, we aimed to investigate the occurrence of carbapenemase genes, analyze the genetic diversity by ERIC-PCR, and examine the most common plasmid incompatibility groups (Incs) in clinical isolates of K. pneumoniae from colonization and infection in patients from a hospital in Brazil. METHODS: Twenty-seven isolates of carbapenem-resistant K. pneumoniae were selected and screened for the presence of carbapenemase genes and Incs by PCR, followed by amplicon sequencing. RESULTS: The bla KPC and bla NDM genes were detected in 24 (88.8 %) and 16 (59.2 %) of the isolates, respectively. Thirteen isolates (48.1 %) were positive for both genes. The IncFIB (92.6 %) and IncQ (88.8 %) were the most frequent plasmids, followed by IncA/C, IncHI1B, and IncL/M, indicating that plasmid variability existed in these isolates. To our knowledge, this is the first report of IncHI1B in Brazil. We found eight isolates with clonal relationship distributed in different sectors of the hospital. CONCLUSIONS: The accumulation of resistance determinants, the variability of plasmid Incs, and the clonal dissemination detected in K. pneumoniae isolates demonstrate their potential for infection, colonization, and the dissemination of different resistance genes and plasmids.


Asunto(s)
Infecciones por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Brasil , Hospitales Públicos , Humanos , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , beta-Lactamasas/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA