Characterization of DNA cleavage produced by seminal plasma using leukocytes as a cell target.

Numerous studies have shown the presence of DNA lesions in human spermatozoa affecting sperm quality. However, the nature of this anomaly and its relationship with patient etiology are poorly understood since different mechanisms can be involved in the formation of these novel DNA configurations including the action of a seminal plasma nuclease activity. The objective of this study was to assess the capacity of seminal plasma for producing endogenous DNA cleavage using nuclei of peripheral blood leukocytes as external targets. For this purpose, we used seminal plasma from fertile males with normal semen parameters to produce DNA cleavage in a sample of leukocytes. Three different tests were performed to visualize DNA cleavage: (a) DNase activity detection, (b) DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH), and (c) Two-dimensional comet assay (Two-tail comet assay). Our results demonstrate that: (i) the seminal plasma is able to cleave DNA compacted with histones in the leukocytes; (ii) this DNA cleavage can be associated with DNase activity and (iii) DNA damage mainly corresponds to single-strand DNA breaks. In conclusion, capacity of seminal plasma for producing DNA cleavage represents a solid contribution to expand the analysis of the standard seminal profile and could constitute a putative diagnostic tool for evaluating male infertility.Abbreviations: ALS: alkali labile sites; ART: Assisted Reproduction Technologies; DBD-FISH: DNA Breakage Detection-Fluorescence In Situ Hybridization; DNA: deoxyribonucleic acid; DSBs-DNA: double-strand DNA; FITC: Fluorescein IsoThioCyanate; GEDA: Gravity Enforced Diffusion Assays; PBS: phosphate-buffered saline; ROS: Reactive Oxigen Species; SSBs-DNA: single-strand DNA; SSC: saline-sodium citrate.

A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity.

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

Oligomer based real-time detection of microorganisms producing nuclease enzymes.

In this study, we provide a method using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases in real time, while growing them in culture media. The detection of such microorganisms was possible in a short period of time, as short as 10 minutes up to a maximum of 8 hours, depending on the bacterial density. We also showed the suitability of this new method for determination of minimum inhibitory concentration (MIC) in culture media in a very short period of time, compared to conventional methods. We believe that it can make a significant contribution to gain new insights for analysis of complex materials such as clinical samples, food samples and environmental samples.

Hairpin loop-enhanced fluorescent copper nanoclusters and application in S1 nuclease detection.

Novel highly fluorescent copper nanoclusters (CuNCs) were prepared by using 24 adenine-thymine pair dsDNA (AT24) with six-base (X6) loops (AT24-X6-hairpin DNA) as an effective template. The AT24 double strand stem serves as a template for CuNC formation, and the six-base sequence loop acts as specific regions to enhance the fluorescence intensity of CuNCs. Relative to the AT24-CuNCs, AT24-X6-hairpin CuNCs have greater fluorescence (5 times enhancement). What's more, the influence of the hairpin loop with different base types and base numbers on the fluorescence of CuNCs was first proposed and investigated. By choosing an AT24 double strand stem, any types of base loops can enhance the fluorescence of CuNCs. However, the fluorescence enhancement would be reduced with an increasing number of hairpin loop sequences. Besides this, the successful detection of S1 nuclease demonstrates its potential to be a new and robust fluorescent probe for sensing applications.

Absence of nuclease activity in commonly used oxygen-scavenging systems.

OBJECTIVE: Oxygen scavenging systems are routinely used during single-molecule imaging experiments to improve fluorescent dye stability. Previous work has shown nuclease contamination in the commonly used oxygen scavenging systems. This study evaluates the potential for nuclease contamination in these oxygen scavenging systems. RESULTS: Linear and plasmid DNA was incubated with two different oxygen scavenging systems (1) protocatechuic acid (PCA)-protocatechuate-3,4-dioxygenase (PCD) and (2) glucose-coupled glucose oxidase/catalase (GODCAT). No nucleic acid degradation was observed on single and double-stranded linear DNA and plasmid DNA, indicating the absence of nuclease contamination in these oxygen scavenging systems.

Molecular signatures of neutrophil extracellular traps in human visceral leishmaniasis.

BACKGROUND: Infections with parasites of the Leishmania donovani complex result in clinical outcomes that range from asymptomatic infection to severe and fatal visceral leishmaniasis (VL). Neutrophils are major players of the immune response against Leishmania, but their contribution to distinct states of infection is unknown. Gene expression data suggest the activation of the NETosis pathway during human visceral leishmaniasis. Thus, we conducted an exploratory study to evaluate NET-related molecules in retrospective sera from VL patients, asymptomatic individuals and uninfected endemic controls. RESULTS: We demonstrate that VL patients and asymptomatic individuals exhibit differential regulation of molecules associated with neutrophil extracellular traps (NET). These differences were observed at the transcriptional level of genes encoding NET-associated proteins; in quantifications of cell free DNA and metalloproteinase 9; and in enzymatic activity of DNAse and elastase. Moreover, multivariate analysis resulted in class-specific signatures, and ROC curves demonstrate the ability of these molecules in discriminating asymptomatic infection from uninfected controls. CONCLUSION: Molecules that are associated with NETs are differentially regulated between distinct states of infection with L. infantum, suggesting that NETs might have distinct roles depending on the clinical status of infection. Although unlikely to be exclusive for VL, these signatures can be useful to better characterize asymptomatic infections in endemic regions of this disease.

Single-Stranded DNA Assisted Cell Penetrating Peptide-DNA Conjugation Strategy for Intracellular Imaging of Nucleases.

Cell penetrating peptides (CPPs) are very useful tools for delivery of DNA molecules into living cells without damaging the cell membranes. However, covalent conjugation of DNAs to CPPs is technically difficult, and the reactions between DNA and target nucleases are also liable to be affected by the cationic CPP molecules. In this work, we demonstrate that the electrostatic interactions between CPPs and single-stranded DNA (ssDNA) were stronger than those between CPP and double-stranded DNA (dsDNA). Taking advantage of this property, we developed an ssDNA protected CPP-DNA fluorescent probe which allowed for noninvasive and efficient cellular uptake and rapid imaging of target nucleases in living cells. The probe is highly sensitive and selective. This work represents the first example of using CPP-DNA conjugate to deliver DNA fluorescent probes for in situ imaging of nucleases within cells. The developed approach also holds great potential for the cellular delivery of other nucleic acid molecules for diagnosis or therapeutics purposes.

Nuclease activity as a specific biomarker for breast cancer.

We report on the activity of nucleases derived from cancer cells as a means for specific targeting using nucleic acid probes (substrates). We hypothesize that cancer cells can be differentiated from healthy cells based on their nuclease activity profile, and thus, any method based on this property represents a novel alternative for diagnostic and therapeutic intervention.

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Construction of Metal-Ion-Free G-quadruplex-Hemin DNAzyme and Its Application in S1 Nuclease Detection.

In this work, a new kind of peroxidase-mimicking DNAzyme (G-quadruplex-hemin DNAzyme, G4-hemin) was constructed by using hemin-modified G-rich DNA (hemin-G-DNA). Experimental results demonstrated that the G-rich DNA can form a G-quadruplex structure by the inducement of terminally modified hemin, rendering the assembly of hemin and G-quadruplex structure spontaneously and efficiently. As a result, G-hemin revealed higher peroxidase activity than traditional G-quadruplex/hemin DNAzyme (G4/hemin). Besides, different from G4/hemin, G4-hemin was constructed in one step without the participation of metal ions and adscititious hemin. Accordingly, the construction procedure was significantly simplified and the background signal from dissociative hemin was remarkably reduced. In a proof-of-concept trial, according to the colorimetric signals of G4-hemin, a novel biosensor for the detection of S1 nuclease activity was established, which provides a novel perspective for designing peroxidase-mimicking DNAzyme-based biosensors.

[Cytochemical localization and properties of selected nucleolytic enzymes]. / Cytochemiczna lokalizacja i wlasciwosci wybranych enzymów nukleolitycznych.

In the article there are shortly outlined studies on cytochemical localization of selected nucleolytic enzymes carried out between 1957-1986 by David Shugar and his coworkers. The histochemical localization of several nucleolytic enzymes in animal and plant tissues was determined by synthesis of specific substrates, alpha-naphthyl esters of 5'- and 3'-nucleotides and their derivatives. In rat tissues phosphodiesterase I was localized in the plasma membrane whereas phosphodiesterase II in the lizosomes, reflecting their physiological roles. The localization of pancreatic type ribonuclease in animal tissues was determined, indicating its role in extracellular digestion. Plant nucleotide pyrophosphatase was localized in several tissues, purified to near homogeneity from potato tubers and its properties and substrate specificity were determined. Application of this enzyme for removal of m7GMP from the "cap" of eukaryotic mRNA allowed to elucidate the role of "cap" in mRNA binding to ribosomes in the process of translation. Furthermore, cyclic nucleotide phosphodiesterase was isolated from potato tubers and its physicochemical properties, oligomeric structure and substrate specificity were elucidated.

DNA sensor's selectivity enhancement and protection from contaminating nucleases due to a hydrated ionic liquid.

The thermodynamic stability of certain mismatched base pairs has made the development of DNA sequence sensing systems challenging. Thus, the stability of fully matched and mismatched DNA oligonucleotides in the hydrated ionic liquid choline dihydrogen phosphate (choline dhp) was investigated. Mismatched base pairs were significantly destabilized in choline dhp relative to those in aqueous buffer. A molecular beacon that forms a triplex with a conserved HIV-1 sequence was then designed and tested in choline dhp. The molecular beacon specifically detected the target duplex via triplex formation at concentrations as low as 1 pmol per 10 µL with 10,000-fold sequence selectivity. Moreover, the molecular beacon was protected from a contaminating nuclease in choline dhp, and DNAs in aqueous solutions were not sufficiently stable for practical use.

Highly sensitive determination of ssDNA and real-time sensing of nuclease activity and inhibition based on the controlled self-assembly of a 9,10-distyrylanthracene probe.

We report here a fluorescent biosensor for highly sensitive determination of single-stranded DNA (ssDNA) with remarkable fluorescence enhancement and label-free sensing of S1 nuclease activity and inhibition in real time based on ssDNA-controlled self-assembly of a 9,10-distyrylanthracene (DSA) probe with the aggregation-induced emission (AIE) property, thereby avoiding a sophisticated fabrication process and aggregation-caused quenching (ACQ) effect. Compared with previous technologies, this assay has some advantages. First, since the DSA probe can be synthesized through a simple and effective synthetic route and the sensing technology adopts the unlabelled ssDNA, this biosensor shows advantages of simplicity and cost efficiency. Besides, for the determination of ssDNA, S1 nuclease, and inhibitor, the DSA-based probe provides high sensitivity and a good linear relationship due to the AIE property. As a result, we determined the DNA 24-mer concentration as low as 150 pM, and we are able to detect ssDNA lengths with a linear range from 6mer to 24mer (R = 0.998) as well as DNA 24-mer concentrations with a linear range from 0 to 200 nM (R = 0.998) and S1 nuclease concentrations with a linear range from 6 to 32 U ml(-1) (R = 0.995), respectively. Moreover, the fluorescent intensity with various concentrations of S1 nuclease becomes highly discriminating after 3-16 min. Thus, it is possible to detect nuclease activity within 3-16 min, which demonstrates another advantage of a quick response of the present biosensor system.

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application.

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 µg ml(-1)), amoxicillin/clavulanic acid (30 µg ml(-1)) and cephalothin (30 µg ml(-1)) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.

[Pathogenicity factors of enterococci of human intestinal microflora].

AIM: Characteristic of pathogenicity factors of enterococci isolated from human feces. MATERIALS AND METHODS: Production of hemolysin, gelatinase and DNase was determined in 161 enterococci cultures. RESULTS: Hemolytic activity detected in 14.9 +/- 2.8% of the studied cultures was the most prevalent characteristic; 22 of 24 hemolytic strains belonged to E. faecalis species. Human erythrocyte lysis was also caused by E. faecium and E. durans cultures (1 strain each). Other pathogenicity factors were detected solely in E. faecalis species members. Enterococci proteolytic activity associated with gelatinase enzyme production manifested on various substrates--both gelatin and milk. This property was detected in 7.5 +/- 2.1% cultures. Deoxyribonuclease was detected in 1 (1.2 +/- 0.9%) E. faecalis strain. A number of E. faecalis cultures possessing hemolytic activity additionally hydrolyzed gelatin (22.7 +/- 8.9% strains) and DNA (4.5 +/- 4.4% isolates). CONCLUSION: Though pathogenicity factors occur in enterococci of intestine microflora relatively rarely, separate cultures with expression of 2 or more pathogenicity factors may be essential in the development of endogenous infections especially in immune compromised patients.

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

[Persistence properties of Corynebacterium non diphtheriae circulating in Rostov-on-Don and Rostov region].

AIM: Characterize persistence properties and antibiotic sensitivity of Corynebacterium non diphtheriae circulating in Rostov-on-Don and Rostov Region. MATERIALS AND METHODS: DNase, anti-immunoglobulin activity, hemagglutinating activity, antagonistic properties and antibiotic sensitivity of Corynebacterium non diphtheriae strains isolated from patients with inflammatory diseases of urogenital tract, pregnant women and individuals undertaking prophylactic examination were studied. RESULTS: Lack of antagonistic interactions of C. non diphtheriae with members of opportunistic microorganisms, high anti-immunoglobulin activity against IgM and IgA, lack of hemagglutinating activity were established. Cefazolin and benzylpenicillin had the highest antibacterial activity against C. non diphtheriae, azithromycin and lincomycin--the lowest. CONCLUSION: Population level of C. non diphtheriae during urogenital tract disease diagnostics is necessary. Determination of their sensitivity to a wide specter of antibacterial preparations is reasonable for optimal selection of therapeutic agents.

Unique X-ray sheet structure of 1,8-bis(imidazolium) anthracene and its application as a fluorescent probe for DNA and DNase.

A new imidazolium anthracene derivative 1 was synthesized, and its unique X-ray crystal structure was examined. In aqueous solutions, probe 1 exhibited a selective fluorescent quenching effect only with DNA among various anions including the nucleotides investigated. This probe was further applied to monitor the activity of DNase.

[The significance of some potentially pathogenic microorganisms in occurrence of food toxicosis. Part 1. S. aureus and staphylococcal enterotoxins].

The data on the nomenclature, classification and properties of staphylococci and staphylococcal enterotoxins produced by them are presented. The analysis of cultural and biochemical properties of 137 strains of staphylococci isolated from raw milk and "Russian" cheese was performed. The high degree of correlation between the ability of S. aureus produce enterotoxins and the presence of enzymes coagulase, thermostable DNase, and other factors of pathogenicity is established.

Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii.

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2 mm) than among environmental strains (average radius of 2.9 mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31 kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans-C. gattii species complex pathogenicity.