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1.
Adv Exp Med Biol ; 1267: 45-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894476

RESUMO

In this chapter, we will focus on ParABS: an apparently simple, three-component system, required for the segregation of bacterial chromosomes and plasmids. We will specifically describe how biophysical measurements combined with physical modeling advanced our understanding of the mechanism of ParABS-mediated complex assembly, segregation and positioning.


Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Posicionamento Cromossômico , DNA Bacteriano/metabolismo , Plasmídeos/metabolismo
2.
PLoS One ; 15(8): e0237474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857767

RESUMO

The effective treatment of carbapenemase-producing Klebsiella pneumoniae infection has been limited and required novel potential agents. Due to the novel drug development crisis, using old antimicrobial agents and combination therapy have been highlighted. This study focused on fosfomycin which inhibits cell wall synthesis and has potential activity on Enterobacteriaceae. We evaluated fosfomycin activity against carbapenemase-producing K. pneumoniae and characterized fosfomycin resistance mechanisms. Fosfomycin revealed effective activity against only 31.8% of carbapenemase-producing K. pneumoniae isolates. The major resistance mechanism was FosA3 production. The co-occurrence of FosA3 overexpression with the mutation of glpT (or loss of glpT) and/or uhpT was mediated high-level resistance (MIC>256 mg/L) to fosfomycin. Moreover, fosA3 silenced in sixteen fosfomycin-susceptible isolates and the plasmid carrying fosA3 of these isolates increased 32- to 64-fold of fosfomycin MICs in Escherichia coli DH5α transformants. The in vitro activity of fosfomycin combination with amikacin by checkerboard assay showed synergism and no interaction in six (16.2%) and sixteen isolates (43.3%), respectively. No antagonism of fosfomycin and amikacin was observed. Notably, the silence of aac (6)'-Ib and aphA6 was observed in amikacin-susceptible isolates. Our study suggests that the combination of fosfomycin and amikacin may be insufficient for the treatment of carbapenemase-producing K. pneumoniae isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Amicacina/farmacologia , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , beta-Lactamases/genética
3.
BMC Infect Dis ; 20(1): 569, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753067

RESUMO

BACKGROUND: HIV-1 produces defective mutants in the process of reproduction. The significance of the mutants has not been well investigated. METHODS: The plasmids of wild type (HIV-1NL4-3) and Env-defective (HIV-1SG3ΔEnv) HIV-1 were co-transfected into HEK293T cells. The progeny virus was collected to infect MT4 cells. The env gene and near-full-length genome (NFLG) of HIV-1 were amplified and sequenced. The phylogenetic diversity, recombinant patterns and hotspots, and the functionality of HIV-1 Env were determined. RESULTS: A total of 42 env genes and 8 NFLGs were successfully amplified and sequenced. Five types of recombinant patterns of env were identified and the same recombinant sites were detected in different patterns. The recombination hotspots were found distributing mainly in conservative regions of env. The recombination between genes of HIV-1NL4-3 and HIV-1SG3Δenv increased the variety of viral quasispecies and resulted in progeny viruses with relative lower infectious ability than that of HIVNL4-3. The defective env genes as well as NFLG could be detected after 20 passages. CONCLUSION: The existence of the defective HIV-1 promotes the phylogenetic evolution of the virus, thus increasing the diversity of virus population. The role of defective genes may be converted from junk genes to useful materials and cannot be neglected in the study of HIV-1 reservoir.


Assuntos
Evolução Molecular , Infecções por HIV/patologia , HIV-1/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Humanos , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinação Genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
4.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética
5.
PLoS Biol ; 18(8): e3000814, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797039

RESUMO

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.


Assuntos
Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Enterococcus faecalis/genética , Transferência Genética Horizontal , Feromônios/genética , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Conjugação Genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Expressão Gênica , Aptidão Genética , Modelos Estatísticos , Feromônios/biossíntese , Plasmídeos/química , Plasmídeos/metabolismo , Percepção de Quorum/genética , Virulência
6.
PLoS One ; 15(7): e0235641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614888

RESUMO

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Salmonella enterica/genética , Sequenciamento Completo do Genoma/métodos , Antibacterianos/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Salmonella enteritidis/classificação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/classificação , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência
7.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(31): 18424-18430, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690674

RESUMO

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.


Assuntos
RNA Fúngico/genética , RNA Guia/genética , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Fúngico/metabolismo , RNA Guia/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
PLoS Pathog ; 16(6): e1008589, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603362

RESUMO

Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-induced sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is found in all KS-lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of KS-spindle cells that have lost KSHV and proliferate autonomously or via paracrine mechanisms. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although infected cells that lose the KSHV-episome prior to tumor formation lose their tumorigenicity, explanted tumor cells that lost the KSHV-episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV tumorigenesis supporting the possibility of hit and run viral-sarcomagenesis. RNA-sequencing and CpG-methylation analysis were performed on KSHV-positive and KSHV-negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV-positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors occurring en exactly the same position, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV-negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V and other proposed mechanism, could be responsible for driving tumorigenesis in the absence of KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of "hit and run" KSHV-sarcomagenesis and point to the existence of selectable KSHV-induced host mutations that may impact AIDS-KS treatment.


Assuntos
Transformação Celular Viral , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8 , Neoplasias Experimentais , Plasmídeos , Sarcoma de Kaposi , Animais , Linhagem Celular , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Plasmídeos/genética , Plasmídeos/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia
10.
PLoS One ; 15(7): e0235532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614905

RESUMO

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.


Assuntos
Centrômero/genética , Colorimetria/métodos , Pichia/genética , Plasmídeos/análise , DNA Fúngico/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
11.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
12.
PLoS One ; 15(5): e0231980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357188

RESUMO

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência
13.
J Vis Exp ; (158)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32364550

RESUMO

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.


Assuntos
Genes Reporter , Recombinação Genética/genética , Genética Reversa/métodos , Rotavirus/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Viral/genética , Análise de Sequência de RNA
14.
PLoS One ; 15(4): e0231393, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352974

RESUMO

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.


Assuntos
Microbiologia de Alimentos , Variação Genética , Listeria monocytogenes/genética , Virulência/genética , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Humanos , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/patologia , Listeriose/transmissão , Tipagem de Sequências Multilocus , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Sorogrupo , Sequenciamento Completo do Genoma
15.
Int J Nanomedicine ; 15: 2633-2646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368045

RESUMO

Objective: The aim of this study is to fabricate functional scaffolds to gene delivery bone morphogenetic protein-2 (BMP-2) plasmid for bone formation in bone tissue engineering. Methods: Dendriplexes (DPs) of generation 4 polyamidoamin (G4-PAMAM)/BMP-2 plasmid were prepared through microfluidic (MF) platform. The physiochemical properties and toxicity of DPs were evaluated by DLS, AFM, FESEM and MTT assay. In order to create a suitable environment for stem cell growth and differentiation, poly-l-lactic acid (PLLA) and poly-l-lactic acid/poly (ethylene oxide) (PLLA/PEO) scaffolds containing hydroxyapatite nanoparticles (HA) and DPs were fabricated by the electrospinning method. The osteogenic potency of the scaffolds on human adipose tissue-derived mesenchymal stem cells (hASCs) was investigated. Results: The results revealed that tuning the physical properties of DPs by adjusting flow parameters in microfluidic platform can easily improve the cell viability compared to conventional bulk mixing method. Also, the result showed that the presence of HA and DPs in PLLA/PEO scaffold enhanced alkaline phosphatase (ALP) activity and increased the amount of deposited Ca, as well as, related to osteogenesis gen markers. Conclusion: This study indicated that on using the MF platform in preparation of DPs and loading them along with HA in PLLA/PEO scaffold, the osteogenic differentiation of hASCs could be tuned.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Osso e Ossos/fisiologia , Durapatita/química , Microfluídica , Nanofibras/química , Poliaminas/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Adesão Celular , Morte Celular , Diferenciação Celular , Proliferação de Células , Forma Celular , DNA/metabolismo , Dendrímeros/química , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Tamanho da Partícula , Plasmídeos/metabolismo , Poliésteres/química , Resistência à Tração
16.
PLoS One ; 15(4): e0232130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353032

RESUMO

The horizontal transfer of plasmids is a key mechanism behind the spread of antibiotic resistance in bacteria. So far, transfer rate constants were measured for a variety of plasmids, donors and recipients. The employed strains typically had a long history in laboratories. Existing data are, therefore, not necessarily representative for real-world environments. Moreover, information on the inter-strain variability of plasmid transfer rates is scarce. Using a high-throughput approach, we studied the uptake of RP4 by various Escherichia coli recipients using Serratia marcescens as the donor. The recipient strains were isolated from human-borne sewage and river sediments. The rate constants of plasmid transfer generally followed a log-normal distribution with considerable variance. The rate constants for good and poor recipients (95 and 5% quantile) differed by more than three orders of magnitude. Specifically, the inter-strain variability of the rate constant was large in comparison to alterations induced by low-level antibiotic exposure. We did not find evidence for diverging efficiencies of plasmid uptake between E. coli recipients of different origin. On average, strains isolated from river bottom sediments were equally efficient in the acquisition of RP4 as isolates extracted from sewage. We conclude that E. coli strains persisting in the aquatic environment and those of direct human origin share a similar intrinsic potential for the conjugative uptake of certain plasmids. In view of the large inter-strain variability, we propose to work towards probabilistic modeling of the environmental spread of antibiotic resistance.


Assuntos
Conjugação Genética/efeitos dos fármacos , Transferência Genética Horizontal/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Genética Horizontal/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Rios , Serratia marcescens/genética , Esgotos
17.
Nat Commun ; 11(1): 2427, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415193

RESUMO

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Oxirredução , Eletroquímica , Eletrodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferricianetos/química , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Piocianina/química , Percepção de Quorum , Regulon , Salmonella enterica/metabolismo , Espectrometria de Fluorescência
18.
Nucleic Acids Res ; 48(11): 6120-6135, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421777

RESUMO

CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.


Assuntos
Adaptação Fisiológica/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , DNA/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Pareamento de Bases , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , DNA Helicases/metabolismo , Mutação , Hibridização de Ácido Nucleico , Plasmídeos/genética , Plasmídeos/metabolismo , Pyrococcus furiosus/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo
19.
Nucleic Acids Res ; 48(11): 6198-6209, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32379323

RESUMO

Group II introns are self-splicing ribozymes and mobile genetic elements. Splicing is required for both expression of the interrupted host gene and intron retromobility. For the pRS01 plasmid-encoded Lactococcus lactis group II intron, Ll.LtrB, splicing enables expression of the intron's host relaxase protein. Relaxase, in turn, initiates horizontal transfer of the conjugative pRS01 plasmid and stimulates retrotransposition of the intron. Little is known about how splicing of bacterial group II introns is influenced by environmental conditions. Here, we show that low temperatures can inhibit Ll.LtrB intron splicing. Whereas autocatalysis is abolished in the cold, splicing is partially restored by the intron-encoded protein (IEP). Structure profiling reveals cold-induced disruptions of key tertiary interactions, suggesting that a kinetic trap prevents the intron RNA from assuming its native state. Interestingly, while reduced levels of transcription and splicing lead to a paucity of excised intron in the cold, levels of relaxase mRNA are maintained, partially due to diminished intron-mediated mRNA targeting, allowing intron spread by conjugal transfer. Taken together, this study demonstrates not only the intrinsic cold sensitivity of group II intron splicing and the role of the IEP for cold-stress adaptation, but also maintenance of horizontal plasmid and intron transfer under cold-shock.


Assuntos
Temperatura Baixa , Conjugação Genética , Íntrons/genética , Lactococcus lactis/genética , Processamento de RNA , Sequência de Bases , Resposta ao Choque Frio , DNA Nucleotidiltransferases/metabolismo , Evolução Molecular , Transferência Genética Horizontal , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Catalítico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroelementos
20.
Anticancer Res ; 40(4): 2025-2032, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234893

RESUMO

BACKGROUND/AIM: The winemaking procedure results in the generation of stems, a by-product that is harmful to the environment. Concomitantly, stems are rich in polyphenols and, hence, they are putatively beneficial for human health. MATERIALS AND METHODS: In this study, the grape stem extracts derived from three native Greek vine varieties, namely Mavrodaphne, Muscat and Rhoditis were examined for their chemical composition and antioxidant and antimutagenic properties using a battery of in vitro biomarkers. RESULTS: All extracts are rich in polyphenols. Moreover, they exhibit potent antioxidant and antimutagenic properties with the extract of Mavrodaphne being the strongest in reducing the DPPH• and O2 -• radicals and the Fe3+ and in protecting plasmid DNA from peroxyl radical-induced oxidative modification. CONCLUSION: Therefore, although they are serious pollutants, grape stems contain phytochemicals with important biological properties and can be used as (ingredients of) bio-functional foods to improve certain aspects of human health.


Assuntos
Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Polifenóis/farmacologia , Vitis/química , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Grécia , Humanos , Oxirredução , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Caules de Planta/química , Plasmídeos/efeitos dos fármacos , Plasmídeos/metabolismo , Polifenóis/química , Espécies Reativas de Oxigênio/metabolismo
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