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1.
Nanoscale ; 13(34): 14538-14551, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34473182

RESUMO

The use of cell-penetrating peptides (CPPs), typically HIV-Tat, to deliver therapeutic genes for cancer treatment is hampered by the inefficient delivery and complicated uptake route of plasmid DNA (pDNA). On the one hand, surface charges, particle size and shape essentially contribute to the endocytosis pathway of Tat/pDNA nanocomplexes, and on the other hand, endogenous cellular factors dominantly determine their intracellular trafficking fate and biological outcome. Recent advances in surfactant-modified nanomaterial and dual molecular imaging technology have offered new opportunities for suicide gene therapy. In this study, we employed the cationic surfactant C16TAB to further condense Tat/pDNA nanocomplexes for improving their delivery efficiency and tested the therapeutic effect of Tat/pDNA/C16TAB (T-P-C) nanoparticles carrying the GCV-converted HSV-ttk suicide gene for ovarian cancer. The cellular endocytosis pathway and underlying signal mechanism of T-P-C nanoparticles were further determined. The obtained T-P-C nanoparticles exhibited a small size, positive surface charge, irregular granular shape and high pDNA encapsulation efficiency. The in vitro experiments showed that T-P-C nanoparticles mainly used the macropinocytosis pathway for uptake in ovarian cancer cells. Their internalization and payload gene expression were controlled by the Arf6 GTPase-dependent, Rab GTPase-activated signal axis. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that T-P-C nanoparticles significantly increased the targeted delivery and suicide gene therapy in a mouse model xenografted with human ovarian cancer. More importantly, Arf6-mediated macropinocytosis remarkably enhanced the delivery efficiency and suicide gene therapy effect of T-P-C nanoparticles. Therefore, these C16TAB-condensed Tat/pDNA nanoparticles combined with the dual molecular imaging strategy provides a novel intracellular delivery platform for high-efficient, precise suicide gene therapy of ovarian cancer.


Assuntos
Nanopartículas , Neoplasias Ovarianas , Animais , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Camundongos , Neoplasias Ovarianas/terapia , Plasmídeos , Transfecção
2.
Mater Sci Eng C Mater Biol Appl ; 128: 112307, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474858

RESUMO

Gene transfer to mesenchymal stem cells (MSCs) has arisen as a powerful approach to increase the therapeutic potential of this effective cell population. Over recent years, niosomes have emerged as self-assembled carriers with promising performance for gene delivery. The aim of our work was to develop effective niosomes-based DNA delivery platforms for targeting MSCs. Niosomes based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA; 0, 7 or 15%) as cationic lipid, cholesterol as helper lipid, and polysorbate 60 as non-ionic surfactant, were prepared using a reverse phase evaporation technique. Niosomes dispersions (filtered or not) and their corresponding nioplexes with a lacZ plasmid were characterized in terms of size, charge, protection, and complexation abilities. DOTMA concentration had a large influence on the physicochemical properties of resulting nioplexes. Transfection efficiency and cytotoxic profiles were assessed in two immortalized cell lines of MSCs. Niosomes formulated with 15% DOTMA provided the highest values of ß-galactosidase activity, being similar to those achieved with Lipofectamine®, but showed less cytotoxicity. Filtration of niosomes dispersions before adding to the cells resulted in a loss of their biological activities. Storage of niosomes formulations (for 30 days at room temperature) caused minor modification of their physicochemical properties but also attenuated the transfection capability of the nioplexes. Differently, addition of the lysosomotropic agent sucrose into the culture medium during transfection or to the formulation itself improved the transfection performance of non-filtered niosomes. Indeed, steam heat-sterilized niosomes prepared in sucrose medium demonstrated transfection capability.


Assuntos
Lipossomos , Células-Tronco Mesenquimais , Técnicas de Transferência de Genes , Humanos , Plasmídeos/genética , Transfecção
3.
BMC Bioinformatics ; 22(1): 390, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34332528

RESUMO

BACKGROUND: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. RESULTS: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. CONCLUSIONS: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.


Assuntos
Transferência Genética Horizontal , Genômica , Resistência Microbiana a Medicamentos , Plasmídeos/genética , Fatores de Virulência
4.
Appl Microbiol Biotechnol ; 105(14-15): 5959-5972, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34357429

RESUMO

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.


Assuntos
Origem de Replicação , Yarrowia , Centrômero , Replicação do DNA , Engenharia Metabólica , Plasmídeos/genética , Yarrowia/genética
5.
Front Cell Infect Microbiol ; 11: 698146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368016

RESUMO

L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.


Assuntos
Arabinose , Proteínas de Bactérias , Biofilmes , Salmonella typhimurium , Arabinose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
6.
Zhonghua Yi Xue Za Zhi ; 101(31): 2478-2484, 2021 Aug 17.
Artigo em Chinês | MEDLINE | ID: mdl-34399563

RESUMO

Objective: To characterize the antibiotic resistance and virulence in a carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A CRKP (designated K. pneumoniae C35) was isolated from a stool sample. The minimal inhibitory concentrations of antimicrobial agents were determined using the broth microdilution method. Whole-genome sequencing and genome analysis were performed to identify the antibiotic resistance and virulence genes. The genetic relationship among K. pneumoniae C35 and other CRKP isolates from our hospital was analyzed by single nucleotide polymorphism (SNP) typing of core genomes. Conjugation experiments were carried out by filter mating to evaluate the transferability and efficiency of resistance genes. The virulence phenotype was determined by Galleria mellonella infection model. Results: K. pneumoniae C35 exhibited resistance to the majority of tested antibiotics, especially carbapenems, sulbactam, and polymyxins. SNP typing showed that K. pneumoniae C35 shared a high degree of sequence homology with several CRKP isolates from different wards. This ST11 CRKP carried 13 resistance genes, including blaKPC-2, blaCTX-M-199, mcr-1, and tet(A) variant. blaKPC-2 gene was located on an IncFⅡ plasmid with>69 800 bp in size, blaCTX-M-199 and mcr-1 genes were located on an IncI2 plasmid (>64 800 bp), and tet(A) variant was located on an unknown Inc-type plasmid (83 628bp). All these three plasmids were conjugative. K. pneumoniae C35 was found to harbor rmpA, rmpA2, and iucABCD aerobactin-related genes, and was considered to be classic carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP). The virulence potential of this strain was confirmed in a Galleria mellonella infection model. The survival rate of the larvae injected with strain C35 at 48 h after infection was significantly lower than that of negative control strain (16.7% vs 80.0%). Conclusion: Multiple conjugative plasmids are identified in a faecal CR-hvKP. The IncI2 plasmid co-carrying both blaCTX-M-199 and mcr-1 genes is firstly identified in CR-hvKP. The emergence of such strain should be alerted and active surveillance is warranted.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Resistência Microbiana a Medicamentos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Tipagem de Sequências Multilocus , Plasmídeos/genética , Virulência/genética , beta-Lactamases
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360889

RESUMO

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.


Assuntos
Genes Virais/imunologia , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Imunidade Celular , Células-Tronco Mesenquimais/imunologia , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Camundongos , Camundongos Endogâmicos DBA , Plasmídeos/genética , Linfócitos T/imunologia , Transfecção , Resultado do Tratamento , Vacinas de DNA/imunologia
8.
Microb Pathog ; 159: 105124, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34364978

RESUMO

OBJECTIVES: Pseudomonas aeruginosa is a medically important pathogen showing intrinsic low permeability to various antimicrobial agents and its potential to acquire multiple resistance mechanism. A longitudinal surveillance aimed to investigate the antimicrobial resistance and its determinants of Pseudomonas aeruginosa in Southern China. A total of 2163 P. aeruginosa isolates were obtained from patients in Southern China during 2004-2016. METHODS: The antimicrobial susceptibility of the isolates was performed by disk diffusion and Vitek 2 automated system and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) 2015. RESULTS: A significant downtrend of resistant rate (>10.0%) was observed for tested antibiotic agents including ciprofloxacin (>30.0%), gentamicin (29.0%), tobramycin (24.2%) and ceftazidime (24.0%) except for aztreonam and amikacin. A total of 269 randomly selected isolates were further studied on the carriage of ß-lactam resistance genes by using 7 groups of multiplex PCRs targeting on 20 genes. ß-lactam resistance genes were rarely detected with a rate lower than 8%. Among all ß-lactam resistance genes, blaSHV acquired the highest identification rate (18/269, 6.7%), followed by blaOXA-1-like (6/269, 2.2%) and blaPER (6/269, 2.2%). In addition, 8 different plasmid replicons were amplified using 8 groups of multiplex PCRs including 18 sets of primers. Only five plasmid replicons were identified in 5 different P. aeruginosa isolates. Insignificant clonal relatedness among the positive strains identified by regular PCR were further verified by randomly amplified polymorphic DNA (RAPD)-PCR. CONCLUSION: This study has provided comprehensive knowledge on current antimicrobial resistance, ß-lactam resistance genes and plasmid replicons carriage in a large scale of clinical P. aeruginosa isolates.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Replicon , beta-Lactamases/genética
9.
Viruses ; 13(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34452443

RESUMO

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Amplamente Neutralizantes/sangue , Linhagem Celular , Coronavirus Humano 229E/imunologia , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/fisiologia , Reações Cruzadas , Humanos , Lentivirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Plasmídeos , SARS-CoV-2/fisiologia , Transfecção , Internalização do Vírus
10.
Clin Lab ; 67(8)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34383410

RESUMO

BACKGROUND: To investigate the epidemics of plasmid-mediated quinolone resistance (PMQR) gene in carbapenem-resistant Enterobacteriaceae (CRE) and the resistance mechanism. METHODS: We collected CRE bacteria isolated clinically between December 2017 and December 2018 for identification and drug sensitivity testing using a VITEK2 Compact Analyzer. Furthermore, genes, including qnrA, qnrB, qnrS, qepA, and acc (6') Ib-cr, were determined through the polymerase chain reaction and sequencing. The hori-zontal transfer of PMQR gene was validated through the plasmid conjugational test. RESULTS: Drug resistance rate of carbapenem-resistant Escherichia coli against quinolones was 100%, while the rate of carbapenem-resistant Klebsiella pneumoniae ranged from 15.56% to 33.33%. The detection rate of acc (6') Ib-cr was the highest (87.72%), followed by qnrB (77.19%) and qnrS (17.54%). Additionally, there were two bacteria carrying the qnrA gene (3.51%), but qepA gene was not isolated from the samples. In total, 84.21% of these bacteria carried 2 or 3 kinds of PMQR genes. Among 8 bacteria with successful plasmid conjugation, PMQR gene transfer was detected in all of them, but with no significant change in the minimum inhibitory concentration of quinolones. CONCLUSIONS: CRE remain sensitive to quinolones in spite of the high detection rate of PMQR gene in this hospital.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae , Quinolonas , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Quinolonas/farmacologia
11.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360567

RESUMO

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.


Assuntos
Antibacterianos/farmacologia , Queijo/microbiologia , Resistência Microbiana a Medicamentos , Eritromicina/farmacologia , Lactobacillales/crescimento & desenvolvimento , Plasmídeos/genética , Animais , Lactobacillales/efeitos dos fármacos , Lactobacillales/isolamento & purificação
12.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
13.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
14.
Appl Microbiol Biotechnol ; 105(18): 6835-6852, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34448898

RESUMO

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3' region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. KEY POINTS: • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.


Assuntos
Gluconobacter oxydans , Gluconobacter , Ácido Acético , Expressão Gênica , Gluconobacter oxydans/genética , Plasmídeos/genética
15.
Nucleic Acids Res ; 49(15): 8732-8742, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365511

RESUMO

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Animais , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Plasmídeos/genética , Deleção de Sequência , Translocação Genética
16.
Front Cell Infect Microbiol ; 11: 658070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34354959

RESUMO

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in bla KPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in bla KPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored bla KPC-2, bla SHV-182 and bla TEM-1B. Compared with KP135194, KP137060 harbored an additional bla NDM-5 positive plasmid. bla NDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that bla KPC-2- and bla NDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a bla NDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Antibacterianos/farmacologia , Compostos Azabicíclicos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Ceftazidima/farmacologia , Combinação de Medicamentos , Escherichia coli/genética , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética
17.
Nat Commun ; 12(1): 4765, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362925

RESUMO

Antibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an 'omics-based' framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as 'current threats' (Rank I; 3%) - already present among pathogens - and 'future threats' (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 'current threat' ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II ('future threats'). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Bactérias/efeitos dos fármacos , Bactérias/genética , Bases de Dados Factuais , Microbioma Gastrointestinal/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Genoma , Humanos , Metagenoma , Plasmídeos
18.
Sheng Li Xue Bao ; 73(4): 559-570, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34405212

RESUMO

Prostaglandins are a class of poly-unsaturated fatty acids-derived bioactive lipids with important physiological function by binding to specific receptors. Prostaglandin receptors lack specific antibodies, which greatly impedes the research on our understanding of the signaling of prostaglandins. The aim of this study was to identify nine mouse lines with amino terminal (-NH2, -N) HA-tagged prostaglandin receptors by using the combination of artificial sperm and CRISPR-Cas9 technology. The guide RNA expression plasmid and labeled targeting vector plasmids were transferred into "artificial sperm cells". The "artificial sperm cells" containing labeled proteins were selected and injected into mouse oocytes, and implanted into pseudopregnant mice to obtain labeled mice. The genomic DNA of the prostaglandin receptor tagged mice was extracted, and the genotypes of mice were detected by PCR method. We also isolated mouse peritoneal macrophages to verify the protein expression of HA-labeled prostaglandin receptor by Western blot. Specific DNA bands were amplified in prostaglandin receptor labeled mice, and specific HA protein bands were detected in macrophage proteins, which was not detected in wild type mice. In summary, we successfully constructed 9 mouse lines with HA-tagged prostaglandin receptors, providing a powerful tool for further study of the pathophysiological functions of prostaglandin signaling both in vivo and in vitro.


Assuntos
RNA Guia , Receptores de Prostaglandina , Animais , Camundongos , Oócitos , Plasmídeos
19.
Front Cell Infect Microbiol ; 11: 701625, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395312

RESUMO

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the "pre-antibiotic era". Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.


Assuntos
Colistina , Proteínas de Escherichia coli , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos
20.
Microb Pathog ; 159: 105154, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34419612

RESUMO

INTRODUCTION: ESBL producing Escherichia coli (E. coli) have spread in the hospital settings. The aims of this study determination of genetic relationship between Environmental E. coli with PFGE typing and investigation of IS element in blaCTX-M gene of these isolates. MATERIALS AND METHODS: A total of 50 E. coli isolates were collected from hospital environmental. The blaCTX-M producing E. coli and IS element of this gene with phylogenetic typing were detected by PCR. The PFGE was performed to detect genetic relationships between this strains. RESULTS: Most of the isolates were from urology wards, other samples were isolated from ICU, surgery and orthopedic ward. The majority of isolates were resistant to cefotaxime and ceftazidime antibiotics and also phosphomycin antibiotic resistant were detected in 10% of isolates. CTX-M gene was detected in 72% of isolates. Moreover, ISEcp1, IS26a, and IS26b were detected upstream of CTX-M in 24%, 8% and 16 of isolates. A phylogroup was the most frequent and PFGE analysis exhibited a diverse distribution of E. coli isolates. CONCLUSIONS: The results demonstrated the existence of CTX-M-producing E. coli in a hospital environment which is a source for drug-resistant strains. In the most of strains, ISEcp1 was located in the upstream of CTX-M gene and Orf477 was found in the downstream. However, in some strains, IS26 was inserted within the ISEcp1element. Our results show that despite the fact that antibiotics of phosphomycin are not used in this hospital, resistance to phosphomycin was observed in the environmental E. coli.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Escherichia coli/genética , Humanos , Filogenia , Plasmídeos , beta-Lactamases/genética
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