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1.
Arch Microbiol ; 204(10): 631, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36121479

RESUMO

Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent insecticidal activities. In the past several decades, scientists have made great efforts to solve its low productivity. However, a lack of understanding of the regulatory network of milbemycin biosynthesis limited the development of high-producing strains using a regulatory rewiring strategy. SARPs (Streptomyces Antibiotic Regulatory Proteins) family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS (the core minimal PKS encoding genes are sbi_06843, sbi_06844, sbi_06845 and sbi_06846) gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR analysis proved that MilR3 positively affected the transcription of specific genes within milbemycin BGC and those from the type II PKS gene cluster. Unlike previous "small" SARP family regulators that played pathway-specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper level regulator in the MilR3-MilR regulatory cascade. This study first illustrated the co-regulatory role of this unique SARP regulator. This greatly enriches our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future.


Assuntos
Regulação Bacteriana da Expressão Gênica , Streptomyces , Antibacterianos/metabolismo , Desoxirribonucleases/genética , Streptomyces/genética , Streptomyces/metabolismo
2.
Cells ; 11(18)2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36139476

RESUMO

Rationale: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives: Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods: Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p = 0.0397) and NETosis (p = 0.0332), and impaired phagocytosis (p = 0.0036) associated with impaired ROS generation (p < 0.0001). The percentage of CD54+ neutrophils (p < 0.001) was significantly increased, while surface expression of CD11b (p = 0.0014) and PD-L1 (p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p = 0.0396) and impaired DNAse activity (p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p = 0.0129). Conclusions: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity.


Assuntos
COVID-19 , Ácidos Nucleicos Livres , Pneumonia , Antígeno B7-H1 , Desoxirribonucleases , Humanos , Interleucina-6/farmacologia , Neutrófilos/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases , RNA Viral , Espécies Reativas de Oxigênio/metabolismo , SARS-CoV-2 , Fator A de Crescimento do Endotélio Vascular/farmacologia
3.
Arch Microbiol ; 204(10): 628, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-36114880

RESUMO

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.


Assuntos
Colicinas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Colicinas/genética , Colicinas/metabolismo , Desoxirribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Óperon
4.
Database (Oxford) ; 20222022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-36094905

RESUMO

The rapid advancement of sequencing technology, including next-generation sequencing (NGS), has greatly improved sequencing efficiency and decreased cost. Consequently, huge amounts of genomic, transcriptomic and epigenetic data concerning cotton species have been generated and released. These large-scale data provide immense opportunities for the study of cotton genomic structure and evolution, population genetic diversity and genome-wide mining of excellent genes for important traits. However, the complexity of NGS data also causes distress, as it cannot be utilized easily. Here, we presented the cotton omics data platform COTTONOMICS (http://cotton.zju.edu.cn/), an easily accessible web database that integrates 32.5 TB of omics data including seven assembled genomes, resequencing data from 1180 allotetraploid cotton accessions and RNA-sequencing (RNA-seq), small RNA-sequencing (smRNA-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), DNase hypersensitive sites sequencing (DNase-seq) and Bisulfite sequencing (BS-seq). COTTONOMICS allows users to employ various search scenarios and retrieve information concerning the cotton genomes, genomic variation (Single nucleotide polymorphisms (SNPs) and Insertion and Deletion (InDels)), gene expression, smRNA expression, epigenetic regulation and quantitative trait locus (QTLs). The user-friendly web interface offers a variety of modules for storing, retrieving, analyzing and visualizing cotton multi-omics data to diverse ends, thereby enabling users to decipher cotton population genetics and identify potential novel genes that influence agronomically beneficial traits. Database URL: http://cotton.zju.edu.cn.


Assuntos
Gerenciamento de Dados , Epigênese Genética , Desoxirribonucleases , Sequenciamento de Nucleotídeos em Larga Escala , RNA
5.
PLoS One ; 17(9): e0271420, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36155485

RESUMO

Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C. acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C. acnes, but a system for evaluating the induction of innate immunity by C. acnes using melanization as an indicator has not yet been established. Here we demonstrated that C. acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus, a gram-positive bacterium identical to C. acnes, does not cause immediate melanization. Even injection of heat-killed C. acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C. acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C. acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C. acnes and that C. acnes peptidoglycans are involved in the induction of innate immunity in silkworms.


Assuntos
Bombyx , Animais , Desoxirribonucleases , Hemolinfa/microbiologia , Humanos , Lisostafina , Melaninas , Monofenol Mono-Oxigenase , Muramidase , Peptidoglicano/farmacologia , Propionibacterium acnes , Ribonucleases
6.
J Extracell Vesicles ; 11(9): e12268, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36149031

RESUMO

Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.


Assuntos
Ácidos Nucleicos Livres , Vesículas Extracelulares , Aloenxertos , Biomarcadores/urina , Ácidos Nucleicos Livres/genética , DNA , Desoxirribonucleases , Humanos , Rim/patologia
7.
Viruses ; 14(9)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36146715

RESUMO

Members of the jingmenviruses group have been found in arthropods and mammals on all continents except Australia and Antarctica. Two viruses of this group were isolated from patients with fever after a tick bite. Using a nested RT-PCR assay targeting a jingmenvirus polymerase gene fragment, we screened ticks collected in seven regions of Russia and found that the abundant jingmenvirus-positive were of Ixodes ricinus species, with the prevalence ranging from 19.8% to 34.3%. In all cases, DNase/RNase treatment suggested that the detected molecule was DNA and subsequent next generation sequencing (NGS) proved that the viral polymerase gene was integrated in the I. ricinus genome. The copy number of the integrated polymerase gene was quantified by qPCR relative to the ITS2 gene and estimated as 1.32 copies per cell. At least three different genetic variants of the integrated polymerase gene were found in the territory of Russia. Phylogenetic analysis of the integrated jingmenvirus polymerase gene showed the highest similarity with the sequence of the correspondent gene obtained in Serbia from I. ricinus.


Assuntos
Ixodes , Animais , Desoxirribonucleases , Humanos , Mamíferos , Filogenia , Reação em Cadeia da Polimerase , Ribonucleases
8.
J Microbiol Biotechnol ; 32(9): 1134-1145, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36116920

RESUMO

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.


Assuntos
Streptomyces coelicolor , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Antraquinonas/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA , Desoxirribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Regiões Promotoras Genéticas , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética
9.
Nat Commun ; 13(1): 5533, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36130957

RESUMO

Genome-wide profiling of chromatin accessibility by DNase-seq or ATAC-seq has been widely used to identify regulatory DNA elements and transcription factor binding sites. However, enzymatic DNA cleavage exhibits intrinsic sequence biases that confound chromatin accessibility profiling data analysis. Existing computational tools are limited in their ability to account for such intrinsic biases and not designed for analyzing single-cell data. Here, we present Simplex Encoded Linear Model for Accessible Chromatin (SELMA), a computational method for systematic estimation of intrinsic cleavage biases from genomic chromatin accessibility profiling data. We demonstrate that SELMA yields accurate and robust bias estimation from both bulk and single-cell DNase-seq and ATAC-seq data. SELMA can utilize internal mitochondrial DNA data to improve bias estimation. We show that transcription factor binding inference from DNase footprints can be improved by incorporating estimated biases using SELMA. Furthermore, we show strong effects of intrinsic biases in single-cell ATAC-seq data, and develop the first single-cell ATAC-seq intrinsic bias correction model to improve cell clustering. SELMA can enhance the performance of existing bioinformatics tools and improve the analysis of both bulk and single-cell chromatin accessibility sequencing data.


Assuntos
Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Cromatina/genética , DNA Mitocondrial , Desoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Lineares , Análise de Sequência de DNA/métodos , Análise de Célula Única , Fatores de Transcrição/metabolismo
10.
Shock ; 58(3): 217-223, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35959777

RESUMO

ABSTRACT: Introduction: Neutrophil extracellular traps (NETs) trigger thrombin generation. We aimed to characterize the effects of deoxyribonuclease (DNAse) on NET components (cell-free DNA [cfDNA] and histones) and thrombin generation after trauma. Methods: Citrated plasma samples were collected from trauma patients and healthy volunteers. Thrombin generation (calibrated automated thrombogram) was measured as lag time (LT, in minutes), peak height (in nM), and time to peak thrombin generation (in minutes). Citrullinated histone 3 (CitH3) and 4 (CitH4) were measured by enzyme-linked immunosorbent assay; cfDNA by PicoGreen (all in nanograms per milliliter). Samples analyzed +/- DNAse (1,000 U/mL). Results expressed as median and quartiles [Q1, Q3], Wilcoxon testing, P < 0.05 significant. Results: We enrolled 46 patients (age, 48 [31, 67] years; 67% male) and 21 volunteers (age, 45 [28, 53] years; 43% male). Deoxyribonuclease treatment of trauma plasma led to shorter LT (3.11 [2.67, 3.52] min; 2.93 [2.67, 3.19] min), shorter time to peak thrombin generation (6.00 [5.30, 6.67] min; 5.48 [5.00, 6.00] min), greater peak height (273.7 [230.7, 300.5] nM; 288.7 [257.6, 319.2] nM), decreased cfDNA (576.9 [503.3, 803.1] ng/mL; 456.0 [393.5, 626.7] ng/mL), decreased CitH3 (4.54 [2.23, 10.01] ng/mL; 3.59 [1.93, 7.98] ng/mL), and increased H4 (1.30 [0.64, 6.36] ng/mL; 1.75 [0.83, 9.67] ng/mL), all P < 0.001. The effect of DNAse was greater on trauma patients as compared with volunteers for LT (ΔLT, -0.21 vs. -0.02 min, P = 0.007), cfDNA (ΔcfDNA -133.4 vs. -84.9 ng/mL, P < 0.001), and CitH3 (ΔCitH3, -0.65 vs. -0.11 ng/mL, P = 0.004). Conclusion: Deoxyribonuclease treatment accelerates thrombin generation kinetics in trauma patient samples as compared with healthy volunteers. These findings suggest that NETs may contribute to the hypercoagulable state observed in trauma patients.


Assuntos
Ácidos Nucleicos Livres , Armadilhas Extracelulares , Desoxirribonucleases , Armadilhas Extracelulares/metabolismo , Feminino , Histonas , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Solubilidade , Trombina/metabolismo
11.
Microb Pathog ; 171: 105715, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35973648

RESUMO

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2″)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2″)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.


Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Aminoglicosídeos , Antibacterianos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/genética , Farmacorresistência Bacteriana/genética , Enterococcus/genética , Enterococcus faecalis , Gelatinases , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Canamicina Quinase/genética , Testes de Sensibilidade Microbiana , Vancomicina , Virulência/genética , Fatores de Virulência/genética
12.
Water Res ; 223: 119009, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36037713

RESUMO

Although multiple experimental studies have proven the use of free synthetic DNA as tracers in hydrological systems, their quantitative fate and transport, especially through the vadose zone, is still not well understood. Here we simulate the water flow and breakthrough of deuterium (D) and one free synthetic DNA tracer from a 10-day experiment conducted in a transient variably saturated 1m3 10° sloped lysimeter using the HYDRUS-2D software package. Recovery and breakthrough flux of D (97.78%) and the DNA tracer (1.05%) were captured well with the advection-dispersion equation (R2 = 0.949, NSE = 0.937) and the Schijven and Simunek two-site kinetic sorption model recommended for virus transport modeling (R2 = 0.824, NSE = 0.823), respectively. The degradation of the DNA tracer was very slow (estimated to be 10% in 10 days), because the "loamy sand" porous media in our lysimeter was freshly crushed basaltic tephra (i.e., crushed rocks) and the microbes and DNase that could potentially degrade DNA in regular soils were rare in our "loamy sand". The timing of the concentration peaks and the HYDRUS-2D simulated temporal and spatial distribution of DNA in the lysimeter both revealed the role of the solid-water-air contact lines in mobilizing and carrying DNA tracer under the experimental variably saturated transient flow condition. The free DNA was nearly non-selectively transported through the porous media, and showed a slightly early breakthrough, possibly due to a slight effect of anion exclusion or size exclusion. Our results indicate that free DNA have the potential to trace vadose zone water flow and solute/contaminant transport, and to serve as surrogates to trace viral pathogen pollution in soil-water systems. To our knowledge, this study is the first to simulate transport mechanisms of free synthetic DNA tracers through real soil textured porous media under variably saturated transient flow condition.


Assuntos
Água Subterrânea , Movimentos da Água , Desoxirribonucleases , Deutério , Modelos Teóricos , Areia , Solo , Água
13.
Viruses ; 14(8)2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-36016404

RESUMO

DNA damage response (DDR) is an evolutionarily conserved mechanism by which eukaryotic cells sense DNA lesions caused by intrinsic and extrinsic stimuli, including virus infection. Although interactions between DNA viruses and DDR have been extensively studied, how RNA viruses, especially coronaviruses, regulate DDR remains unknown. A previous study showed that the porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the Coronaviridae family, induces DDR in infected cells. However, the underlying mechanism was unclear. This study showed that PEDV activates the ATM-Chk2 signaling, while inhibition of ATM or Chk2 dampens the early stage of PEDV infection. Additionally, we found that PEDV-activated ATM signaling correlates with intracellular ROS production. Interestingly, we showed that, unlike the typical γH2AX foci, PEDV infection leads to a unique γH2AX staining pattern, including phase I (nuclear ring staining), II (pan-nuclear staining), and III (co-staining with apoptotic bodies), which highly resembles the apoptosis process. Furthermore, we demonstrated that PEDV-induced H2AX phosphorylation depends on the activation of caspase-7 and caspase-activated DNAse (CAD), but not ATM-Chk2. Finally, we showed that the knockdown of H2AX attenuates PEDV replication. Taken together, we conclude that PEDV induces DDR through the ROS-ATM and caspase7-CAD-γH2AX signaling pathways to foster its early replication.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Animais , Infecções por Coronavirus/veterinária , Desoxirribonucleases , Fosforilação , Vírus da Diarreia Epidêmica Suína/genética , Espécies Reativas de Oxigênio , Transdução de Sinais , Suínos
14.
Int J Mol Sci ; 23(15)2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35955509

RESUMO

TatD960 and TatD825 are DNases that contribute to biofilm formation and virulence in Trueperella pyogenes (T. pyogenes). Luteolin is a natural flavonoid commonly found in plants that exhibits antimicrobial capacity. Our study aims to investigate the effects of luteolin on TatD DNases as a natural inhibitor. In this research, the expression of tatD genes and TatD proteins in T. pyogenes treated with luteolin was detected, and then the effect of luteolin on the hydrolysis of DNA by TatD DNases was analyzed using agarose gel electrophoresis. Moreover, the interactions between luteolin and TatD DNases were tested using surface plasmon resonance (SPR) assays and molecular docking analysis. After 1/2 MIC luteolin treatment, the transcription of tatD genes and expression of TatD proteins appeared to be reduced in 80-90% of T. pyogenes (n = 20). The gel assay revealed that luteolin can inhibit the activity of TatD DNases. The SPR assay showed that the KD values of luteolin to TatD960 and TatD825 were 6.268 × 10-6 M and 5.654 × 10-6 M, respectively. We found through molecular docking that hydrogen bonding is predominant in the interaction of luteolin and TatD DNases. Our data indicate that luteolin inhibited the ability of TatD DNases by decreasing their binding to DNA. The current study provides an insight into the development of luteolin as a DNase inhibitor in preventing biofilm formation and virulence in T. pyogenes.


Assuntos
Desoxirribonucleases , Luteolina , Desoxirribonucleases/metabolismo , Luteolina/farmacologia , Simulação de Acoplamento Molecular , Virulência , Fatores de Virulência/genética
15.
Vet Microbiol ; 273: 109529, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35944391

RESUMO

Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.


Assuntos
Desoxirribonucleases , Armadilhas Extracelulares , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Macrófagos/microbiologia , Camundongos , Mycobacterium avium subsp. paratuberculosis/enzimologia , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Paratuberculose/microbiologia , Virulência
16.
Pulm Pharmacol Ther ; 76: 102146, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35868601

RESUMO

OBJECTIVES: Compare the use of Tissue Plasminogen Activator (t-PA) and t-PA + Dornase (DNase) for the management of complicated pleural effusions, and to determine if a dose-response relationship exists for t-PA. METHODS: Retrospective cohort study that examined all adult patients at a large academic medical center who received intrapleural t-PA or t-PA + DNase for the management of a complicated pleural effusions. Outcomes were success of therapy [defined as avoidance of secondary interventions (i.e. VATSD or thoracotomy)], chest tube output pre- and post-administration, radiographic findings, t-PA dose and frequency, and bleeding complications. RESULTS: Thirty-five patients were enrolled: 25 received t-PA and 10 received t-PA + DNase. Successful pharmacologic treatment occurred in 88% of patients receiving t-PA and 100% of patients receiving t-PA + DNase (p = 0.54). In the t-PA group, chest tube output increased from 75 ml/12 h to 538 ml/12 h after administration of t-PA (p = 0.001), and from 103 ml/12 h to 502 ml/12 h (p = 0.001) in the t-PA + DNase group. Radiographic improvement occurred in 84% of t-PA patients and 90% of t-PA + DNase patients (p = 0.99). In the t-PA group, a successful response occurred in 92% of patients receiving a cumulative dose of ≤10 mg (n = 13) and 83% of patients receiving a cumulative dose of >10 mg (n = 12), p = 0.43. Patients who received a single t-PA dose compared to those who received multiple doses also had similar success rates (p = 1). There was one instance of bleeding following drug administration. CONCLUSION: Both t-PA and t-PA + DNase were highly effective for reducing a patient's need for surgical intervention. Higher cumulative doses or more frequency administrations did not appear to provide additional benefit.


Assuntos
Desoxirribonucleases , Fibrinolíticos , Derrame Pleural , Ativador de Plasminogênio Tecidual , Adulto , Desoxirribonucleases/uso terapêutico , Fibrinolíticos/uso terapêutico , Humanos , Derrame Pleural/tratamento farmacológico , Estudos Retrospectivos , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/uso terapêutico
17.
Mol Cell Probes ; 65: 101844, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35803442

RESUMO

Deoxyribonucleases (DNases) are enzymes that cleave DNA. Some DNases are secreted outside of cells where they can cleave extracellular DNA (ecDNA). High concentration of ecDNA is associated with diseases such as sepsis, preeclampsia, and systemic lupus. DNA can be released from dying cells and is immunogenic. DNases cleave ecDNA and might prevent activation of the immune system. Low DNase activity could be disadvantageous in diseases where high amounts of ecDNA are released from dying cells. The relationship between DNase activity and ecDNA remains unknown. The lack of standard values in DNase activity makes the studies difficult to compare. This review focuses on summarizing methods for DNase activity measurements, the possible implication of decreased DNase activity in diseases, and the impact on diseases associated with a high concentration of ecDNA.


Assuntos
Desoxirribonucleases , Sepse , DNA , Feminino , Humanos , Gravidez
18.
Int J Mol Sci ; 23(14)2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35887361

RESUMO

Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1.


Assuntos
Reparo do DNA , Endonucleases , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonucleases/metabolismo , Endonucleases/metabolismo , Humanos , Proteínas Nucleares/genética , Ligação Proteica
19.
Biosens Bioelectron ; 213: 114475, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-35714494

RESUMO

DNases are enzymes that cleave phosphodiesteric bonds of deoxyribonucleic acid molecules and are found everywhere in nature, especially in bodily fluids, i.e., saliva, blood, or sweat. Rapid and sensitive detection of DNase activity is highly important for quality control in the pharmaceutical and biotechnology industries. For clinical diagnostics, recent reports indicate that increased DNase activity could be related to various diseases, such as cancers. In this paper, we report a new bioelectronic device for the determination of nuclease activity in various fluids. The system consists of a sensor electrode, a custom design DNA target to maximize the DNase cleavage rate, a signal analysis algorithm, and supporting electronics. The developed sensor enables the determination of DNase activity in the range of 3.4 × 10-4 - 3.0 × 10-2 U mL-1 with a limit of detection of up to 3.4 × 10-4 U mL-1. The sensor was tested by measuring nuclease activity in real human saliva samples and found to demonstrate high accuracy and reproducibility compared to the industry standard DNaseAlert™ï¸. Finally, the entire detection system was implemented as a prototype device system utilizing single-use electrodes, custom-made cells, and electronics. The developed technology can improve nuclease quality control processes in the pharmaceutical/biotechnology industry and provide new insights into the importance of nucleases for medical applications.


Assuntos
Técnicas Biossensoriais , Desoxirribonuclease I , Desoxirribonucleases , Humanos , Preparações Farmacêuticas , Reprodutibilidade dos Testes
20.
Int J Mol Sci ; 23(12)2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35743119

RESUMO

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.


Assuntos
Pepinos-do-Mar , Animais , Desoxirribonucleases/metabolismo , Endopeptidases/metabolismo , Humanos , Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Pepinos-do-Mar/metabolismo
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