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1.
Nucleic Acids Res ; 49(13): 7628-7643, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197611

RESUMO

Many type III CRISPR-Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.


Assuntos
Sistemas CRISPR-Cas , Desoxirribonucleases/metabolismo , Transcrição Genética , Trifosfato de Adenosina/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Clivagem do DNA , DNA de Cadeia Simples/metabolismo , Ligantes , Plasmídeos/metabolismo , RNA/análise
2.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
3.
J Photochem Photobiol B ; 221: 112237, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34116318

RESUMO

Nannochloropsis oceanica is widely used as a model photosynthetic chassis to produce fatty acids and carotenoid pigments. However, intense light typically causes excessive generation of reactive oxygen species (ROS) and photorespiration in microalgal cells, which results in decreased cell growth rate and unsaturated fatty acid content. In this study, the Vitreoscilla hemoglobin gene (vgb) was introduced into N. oceanica cells and expressed by using the light-harvesting complex promoter and its signal peptide. Compared with wild type (WT), the growth rate of transformants increased by 7.4%-18.5%, and the eicosapentaenoic acid content in an optimal transformant increased by 21.0%. Correspondingly, the intracellular ROS levels decreased by 56.9%-70.0%, and the catalase content in transformants was about 1.8 times that of WT. The photorespiration level of transformants was reduced by the measurement and calculation of the dissolved oxygen concentration under the condition of light-dark transition. The expression level of the key genes related to the photorespiration pathway in transformants was more than 80% lower than that in WT. These results indicated that Vitreoscilla hemoglobin could improve microalgal growth by reducing ROS damage and modulating photorespiration under stress conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Luz , Estramenópilas/metabolismo , Hemoglobinas Truncadas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Catalase/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Fotossíntese/efeitos da radiação , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Estramenópilas/efeitos da radiação , Hemoglobinas Truncadas/genética
4.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
5.
Gene ; 793: 145745, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34077774

RESUMO

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.


Assuntos
Aspergillus oryzae/enzimologia , Ácidos Graxos/biossíntese , Proteínas Fúngicas/genética , Lipase/genética , Esporos Fúngicos/enzimologia , Triglicerídeos/biossíntese , Aspergillus oryzae/classificação , Aspergillus oryzae/genética , Sistemas CRISPR-Cas , Ácidos Graxos/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Humanos , Microbiologia Industrial , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lipase/metabolismo , Metabolismo dos Lipídeos/genética , Micélio/enzimologia , Micélio/genética , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Triglicerídeos/genética
6.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068033

RESUMO

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK-a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.


Assuntos
Proteínas de Bactérias/metabolismo , Conjugação Genética , DNA Bacteriano/genética , Endodesoxirribonucleases/metabolismo , Klebsiella pneumoniae/enzimologia , Plasmídeos/genética , Recombinação Genética , Proteínas de Bactérias/genética , Sequência de Bases , Endodesoxirribonucleases/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crescimento & desenvolvimento , Plasmídeos/metabolismo
7.
Mol Biochem Parasitol ; 244: 111384, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051228

RESUMO

A tetracycline-responsive transcription system (Tet-Off) adapted for use in Toxoplasma gondii (nicknamed TATi) is useful for molecular biological studies of this organism. Previous studies using TATi incorporated minimal promoters derived from the gene promoters for TgSAG1 or TgSAG4. The present study achieves improved activation and suppression of an integrated reporter gene in the absence and presence of anhydrotetracycline, respectively (p < 0.0001), by use of a newly derived minimal promoter based on the core promoter of TgGRA2. In comparison with the SAG1 minimal promoter, use of the GRA2 minimal promoter in stable transfectants has a 23-fold higher Signal to Noise Ratio for EYFP fluorescence in the absence or presence of anhydrotetracycline. We conclude that the performance of TATi for both activation and suppression of transcription can be markedly enhanced by incorporating a GRA2 minimal promoter.


Assuntos
Antígenos de Protozoários/genética , Proteínas de Bactérias/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Tetraciclinas/farmacologia , Toxoplasma/efeitos dos fármacos , Antígenos de Protozoários/metabolismo , Proteínas de Bactérias/metabolismo , Células Cultivadas , Clonagem Molecular/métodos , Fibroblastos/parasitologia , Expressão Gênica , Genes Reporter , Humanos , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Transcrição Genética , Transfecção
8.
J Biol Chem ; 297(1): 100838, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051235

RESUMO

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.


Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Células COS , Chlorocebus aethiops , Elementos de DNA Transponíveis/genética , Expressão Gênica , Engenharia Genética , Células HEK293 , Humanos , Plasmídeos/metabolismo , Transcrição Genética , Transgenes
9.
Life Sci ; 278: 119570, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33964295

RESUMO

AIMS: Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis. MAIN METHODS: Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology. KEY FINDINGS: EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1f/f mice. SIGNIFICANCE: This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.


Assuntos
Proteínas de Transporte/metabolismo , Endocitose , Exossomos/metabolismo , Regulação da Expressão Gênica , Fator Esteroidogênico 1/metabolismo , Testosterona/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Imunoprecipitação da Cromatina , Feminino , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Fator Esteroidogênico 1/genética , Testosterona/farmacologia
10.
FEBS Lett ; 595(14): 1863-1875, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34032285

RESUMO

Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.


Assuntos
Quimiocina CXCL12/metabolismo , Receptores Adrenérgicos alfa 1/química , Receptores CXCR4/química , Receptores CXCR/química , Receptores de Vasopressinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/farmacologia , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Cinética , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Nat Commun ; 12(1): 2711, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976199

RESUMO

Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.


Assuntos
Edição de Genes/métodos , Redes Reguladoras de Genes , Engenharia Genética/métodos , Genoma Humano , Plasmídeos/metabolismo , Transposases/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Carcinogênese/metabolismo , Diferenciação Celular , Loci Gênicos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Plasmídeos/química , RNA Guia/genética , RNA Guia/metabolismo , Transcrição Genética , Transposases/metabolismo
12.
FEBS J ; 288(10): 3164-3185, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33830641

RESUMO

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.


Assuntos
Apresentação do Antígeno/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Macroautofagia/genética , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Brefeldina A/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macroautofagia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção
13.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33913550

RESUMO

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Assuntos
Proteína DEAD-box 58/imunologia , Evasão da Resposta Imune/genética , Interferons/imunologia , Nucleotidiltransferases/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2/imunologia , Receptor 3 Toll-Like/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Interferons/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Receptores Imunológicos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/genética , Transfecção , Células Vero , Replicação Viral/imunologia
14.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806461

RESUMO

The present study aimed to synthesize novel polycationic polymers composed of N-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)n-O2Oc-NH2, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]8-O2Oc-NH2 and typical for large proteins.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Ácidos Nucleicos/metabolismo , Polímeros/farmacologia , beta-Alanina/análogos & derivados , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Nanoestruturas/química , Plasmídeos/metabolismo , Transfecção/métodos , beta-Alanina/farmacologia
15.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805602

RESUMO

Carriers of genetic material are divided into vectors of viral and non-viral origin. Viral carriers are already successfully used in experimental gene therapies, but despite advantages such as their high transfection efficiency and the wide knowledge of their practical potential, the remaining disadvantages, namely, their low capacity and complex manufacturing process, based on biological systems, are major limitations prior to their broad implementation in the clinical setting. The application of non-viral carriers in gene therapy is one of the available approaches. Poly(amidoamine) (PAMAM) dendrimers are repetitively branched, three-dimensional molecules, made of amide and amine subunits, possessing unique physiochemical properties. Surface and internal modifications improve their physicochemical properties, enabling the increase in cellular specificity and transfection efficiency and a reduction in cytotoxicity toward healthy cells. During the last 10 years of research on PAMAM dendrimers, three modification strategies have commonly been used: (1) surface modification with functional groups; (2) hybrid vector formation; (3) creation of supramolecular self-assemblies. This review describes and summarizes recent studies exploring the development of PAMAM dendrimers in anticancer gene therapies, evaluating the advantages and disadvantages of the modification approaches and the nanomedicine regulatory issues preventing their translation into the clinical setting, and highlighting important areas for further development and possible steps that seem promising in terms of development of PAMAM as a carrier of genetic material.


Assuntos
Dendrímeros/síntese química , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/síntese química , Dendrímeros/administração & dosagem , Regulamentação Governamental , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/metabolismo , Nanomedicina/legislação & jurisprudência , Nanomedicina/métodos , Nanopartículas/administração & dosagem , Nanopartículas/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Propriedades de Superfície
16.
Methods Mol Biol ; 2281: 81-91, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847953

RESUMO

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Sítios de Ligação , DNA Helicases/genética , Replicação do DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
17.
Methods Mol Biol ; 2285: 255-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928558

RESUMO

The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Plasmídeos/genética , Ribonucleoproteínas/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Eletroporação , Ativação Linfocitária , Camundongos , Fenótipo , Plasmídeos/metabolismo , Cultura Primária de Células , Projetos de Pesquisa , Ribonucleoproteínas/metabolismo , Fluxo de Trabalho
18.
Int J Food Microbiol ; 346: 109164, 2021 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-33813365

RESUMO

The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4-8 µg/mL. All mcr-3 gene-positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30-390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, ß-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human.


Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Carne/microbiologia , Alimentos Crus/microbiologia , beta-Lactamases/genética , Animais , Galinhas , Farmacorresistência Bacteriana , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peixes , Contaminação de Alimentos/análise , Contaminação de Alimentos/estatística & dados numéricos , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Prevalência , Alimentos Crus/economia , Suínos , Vietnã , beta-Lactamases/metabolismo
19.
Angew Chem Int Ed Engl ; 60(24): 13536-13541, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33768597

RESUMO

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.


Assuntos
Aminoglicosídeos/metabolismo , Engenharia Genética , Imunossupressores/síntese química , Alquil e Aril Transferases/genética , Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunossupressores/química , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Terpenos/química
20.
Nat Microbiol ; 6(5): 606-616, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33782584

RESUMO

Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterize the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a 2-yr period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonized patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.


Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Carbapenêmicos/farmacologia , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/genética , Microbioma Gastrointestinal , Transferência Genética Horizontal , Plasmídeos/genética , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/terapia , Feminino , Hospitalização , Hospitais Universitários , Humanos , Masculino , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo
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