Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

Isolation, characterization of Weissella confusa and Lactococcus lactis from different milk sources and determination of probiotic features.

This study aimed to investigate the probiotic properties of Lactic Acid Bacteria (LAB) isolates derived from various milk sources. These isolates identified based on their morphological characteristics and 16S rRNA gene sequencing. Four strains of Lactococcus lactis and two strains of Weissella confusa were identified with over 96% 16S rRNA gene similarity according to the NCBI-BLAST results. The survival of the isolates was determined in low pH, pepsin, bile salts, and pancreatin, and their adhesion ability was assessed by in vitro cell adhesion assay, hydrophobicity, auto- and co-aggregation, and safety criteria were determined by hemolytic, gelatinase activities, and DNAse production ability tests. The results showed that the LAB isolates had different levels of resistance to various stress factors. L. lactis subsp. cremoris MH31 showed the highest resistance to bile salt, while the highest pH resistance was observed in L. lactis MH31 at pH 3.0. All the isolates survived in pepsin exposure at pH 3.0 for 3 h. The auto-aggregation test results showed that all strains exhibited auto-aggregation ranging from 84.9 to 91.4%. Co-aggregation percentage ranged from 19 - 54% and 17 - 57% against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. The hydrophobicity capacity of the LAB isolated ranged from 35-61%. These isolates showed different adhesion abilities to Caco-2 cells (81.5% to 92.6%). None of the isolates exhibited DNase, gelatinase and hemolytic activity (γ-hemolysis). All results indicate that these LAB strains have the potential to be used as probiotics.

Intrapleural Thrombolytics as First Line Therapy for Complicated Parapneumonic Effusions and Empyema in Patients with Prohibitive Surgical Risk: A Cases Series.

OBJECTIVE: In complicated parapneumonic effusion or Empyema, approximately 25% of patients require surgical intervention which can be associated with a mortality risk of almost 20%. However, the use combination of rt-tPA and DNase in elderly patients with prohibitive surgical risk has improved outcomes. The main goal of our study is to highlight the utility of intrapleural thrombolysis in patients with prohibitive risk for surgery. METHODS: A retrospective record review study of patients (n=23) with complicated parapneumonic pleural effusion or empyema treated with tPA and DNase from January 1st of 2015 to March 18th, 2019 at VACHCS. Data collected to describe the outcome of intrapleural thrombolytics included demographic, pleural fluid analysis, surgical risk assessment, diagnosis and initiation treatment day, doses, chest imaging, drainage rate, chest tube size and average days in place, inflammatory markers, microbiology, antibiotics, and complications. RESULTS: Only 21.7% of patients were considered surgical candidates. Seventy-four percent had a 30-day post-surgical mortality risk of > 2.5% using the National Surgery Office (NSO) risk calculator. Post-operative inpatient stay was 99.7% and estimated post operative ICU stay average was >80%. Primary outcome (pleural drainage improvement) obtained in 73.9%. Most common serious complications included sepsis (52.2%) and nonserious was residual hydropneumothorax (47.8%). CONCLUSION: This study demonstrates that administration of intrapleural thrombolytics through a percutaneous pleural catheter achieved successful drainage safely and without the need for surgical interventions in a selected group of advanced age, elderly patients with pleural infections who were deemed to be high surgical risk.

DNase enhances photodynamic therapy against fluconazole-resistant Candida albicans biofilms.

OBJECTIVE: This study evaluated the effectiveness of DNase I combined with antimicrobial photodynamic therapy, mediated by Photodithazine® and light-emitting diode light, against biofilms formed by a fluconazole-resistant Candida albicans strain (ATCC 96901) and two clinical isolates (R14 and R70). MATERIALS AND METHODS: Biofilms were grown for 48 h and exposed to DNase for 5 min, followed by application of a photosensitizer (P) and light (L), either singly or combined (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase, and P-L+DNase; n = 12). Biofilm analysis included quantification of extracellular matrix components (water-soluble and insoluble polysaccharides, proteins and extracellular DNA), and biomass (total and insoluble), as well as the enumeration of colony-forming units. The data were analyzed using three-way analysis of variance with Bonferroni's post hoc test. RESULTS: The DNase treatment combined with aPDT showed a reduction of 1.92, 1.65, and 1.29 log10 of cell viability compared with untreated controls for ATCC 96901, R14, and R70 strains, respectively. It also reduced extracellular matrix contents of water-soluble polysaccharides (36.3%) and extracellular DNA (72.3%), as well as insoluble biomass content (43.3%). CONCLUSION: The three strains showed similar behavior when treated with DNase, and the extracellular matrix components were affected, improving the effectiveness of antimicrobial photodynamic therapy.

Heterologous extracellular DNA facilitates the development of Trichosporon asahii and T. inkin biofilms and enhances their tolerance to antifungals.

Trichosporon asahii and T. inkin are emergent agents of deep-seated and disseminated infections in immunocompromised patients. The present study aimed to investigate the role of extracellular DNA (eDNA) and the enzyme deoxyribonuclease (DNase) on the structure of T. asahii and T. inkin biofilms, as well as to examine their effect on the susceptibility to antifungals. Biofilms reached maturity at 48 h; eDNA concentration in the supernatant increased over time (6 < 24 h < 48h). Exogenous eDNA increased biomass of Trichosporon biofilms at all stages of development, enhanced their tolerance to antifungals and improved their structural complexity. DNase reduced biomass, biovolume and thickness of Trichosporon biofilms, thereby rendering them more susceptibility to voriconazole. The results suggest the relevance of eDNA in the structure and antifungal susceptibility of Trichosporon biofilms and highlight the potential of DNase as adjuvant in biofilm control.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

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.

A heterotrinuclear bioinspired coordination complex capable of binding to DNA and emulation of nuclease activity.

The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we demonstrate that a trinuclear coordination complex based on a dinuclear Fe(III)Zn(II) core designed for biomimicry of the hydrolytic enzyme kidney bean purple acid phosphatase, containing an additional pendant arm coordinating a Pd(II) ion, has the ability to interact with DNA and to promote its hydrolytic cleavage. These results were found through analysis of plasmid DNA interaction and cleavage by the trinuclear complex 1 and its derivatives 2 and 3, in addition to the analysis of alteration in the DNA structure in the presence of the complexes through circular dichroism and DNA footprinting techniques. The suggested covalent interaction of the palladium-containing complex with DNA was analysed using an electrophoretic mobility assay, circular dichroism, high resolution gel separation techniques and kinetic analysis. This is a new and promising metal complex targeted to nucleic acids and acting in two separate ways: strong DNA interaction and hydrolytic cleavage.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2',3'-cGAMP-degrading enzyme family.

Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape.

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite.

OBJECTIVE: This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). METHODOLOGY: E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. RESULTS: CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). CONCLUSIONS: Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

In vitro lytic activity and antifungal susceptibility of infrequently isolated yeasts.

Non-albicans Candida species have acquired relevance in the last decades as a cause of serious disease. The virulence factors and antifungal susceptibility of these rare pathogens remain largely unrecognized. We examined a total of 50 yeast isolates corresponding to 11 different infrequently isolated yeast species for their in vitro enzymatic profile and susceptibility pattern as first-line antifungals. We found aspartyl protease activity for 100% of the isolates tested as well as variable DNAse, hemolysin, phospholipase and esterase activities. All strains had low MICs for amphotericin B and showed a variable response to fluconazole (0.125-32 µg/mL) and the echinocandins tested (0.25-> 8 µg/mL).

DNase increases the efficacy of antimicrobial photodynamic therapy on Candida albicans biofilms.

Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes ß-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5 min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5 min, followed by incubation with PDZ for 20 min and exposure to LED light (660 nm, 50 J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZ + DNase, light only, light + DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (p = 0.000) and eDNA amounts (p ≤ 0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT.

Cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps.

Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3'-nucleotidase/nuclease (3'NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3'NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3'NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.

Alteplase and DNase for the treatment of pleural empyema in rats.

BACKGROUND AND OBJECTIVES: Adjunctive intrapleural fibrinolytic is an option to treat empyema at fibrinopurulent stage, but there is controversy about which should be use. Our objective is to evaluate the action of alteplase and/or desoxyribonuclease at physical and chemical properties in vitro pus derived from an experimental induced empyema in rats. METHODS: Streptococcus pneumoniae was introduced into the pleural cavity by thoracentesis through pleural pressure monitor. Animals were euthanized after 24 h, with macroscopic thoracic evaluation and measurement of amount of intrapleural liquid that was posteriorly stored at -80 °C. Selected samples were randomly distributed into four groups, then thawed at room temperature before exposure to one of the following: G1 = alteplase (n = 12), G2 = DNase (n = 12), G3 = alteplase + DNase (n = 12), or G4 = saline (n = 6). The mean molecular size in the fluid portion of the empyema was evaluated using dynamic light scattering; viscosity of the empyema fluid was measured using the drip method. RESULTS: Macroscopic showed purulent liquid, with fibrin and septation, with mean volume of 4.16 ml (0.5-8 ml). All samples were culture-positive for Streptococcus pneumoniae. Comparing with control, all experimental groups presented reduction of larger than 135 nm molecular size, but there was only significant difference with alteplase (p = 0,02). Viscosity reduced at all experimental groups, but increased at control. DNase group presented negative median (-5 mPa/s) of viscosity, and differed significantly from that observed in the control group (p = 0.04). CONCLUSIONS: Alteplase, DNase and alteplase + DNase changed significantly physical and chemical properties of experimental empyema at fibrinopurulent phase: alteplase reduced molecular size larger than 135 nm and DNase reduced viscosity.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Biochemical characterization of the nuclease StoNurA from the hyperthermophilic archaeon Sulfolobus tokodaii.

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.

Sulfonamide-containing copper(II) metallonucleases: Correlations with in vitro antimycobacterial and antiproliferative activities.

The bis-(1,10-phenanthroline)copper(I) complex, [Cu(I)(phen)2]+, was the first copper-based artificial nuclease reported in the literature. The biological and ligand-like properties of sulfonamides make them good candidates for fine-tuning the reactivity of the [Cu(phen)2] motif with biomolecules. In this context, we developed three novel copper(II) complexes containing the sulfonamides sulfameter (smtrH) and sulfadimethoxine (sdmxH) and (N^N)-bidentate ligands (2,2'-biyridine or 1,10-phenantroline). The compounds were characterized by chemical and spectroscopic techniques and single-crystal X-ray crystallography. When targeting plasmid DNA, the phen-containing compounds [Cu(smtr-)2(phen)] (1) and [Cu(sdmx-)2(phen)] (2) demonstrated nuclease activity even in the absence of reducing agents. Addition of ascorbic acid resulted in a complete cleavage of DNA by 1 and 2 at concentrations higher than 10 µM. Experiments designed to evaluate the copper intermediates involved in the nuclease effect after reaction with ascorbic acid identified at least the [Cu(I)(N^N)2]+, [Cu(I)(sulfa)(N^N)]+ and [Cu(I)(sulfa)2]+ species. The compounds interact with DNA via groove binding and intercalation as verified by fluorescence spectroscopy, circular dichroism (CD) and molecular docking. The magnitude and preferred mode of binding are dependent on the nature of both N^N ligand and the sulfonamide. The potent nuclease activity of compounds 1 and 2 are well correlated with their antiproliferative and anti-M. tuberculosis profiles. The results presented here demonstrated the potential for further development of copper(II)-sulfonamide-(N^N) complexes as multipurpose metallodrugs.

Magnetic nanoparticles as a support for a copper (II) complex with nuclease activity.

Magnetic nanoparticles have been extensively explored for the development of platforms for drug delivery and imaging probes. In this work, we have used a modular capping strategy to produce magnetic gold-coated Fe3O4 (Fe3O4@Au) nanoparticles, which have been decorated with a copper (II) complex containing a thioether derivative of clip-phen (Fe3O4@Au@Cu), where the complex [Cu(2CP-Bz-SMe)]2+ has affinity to bind DNA and proven nuclease activity (2CP-Bz-SMe=1,3-bis((1,10-phenanthrolin-2-yl)oxy)-N-(4-(methylthio)benzylidene)propan-2-imine). The functionalization of Fe3O4@Au with the copper complex occurs through the sulfur atom of the thioether moiety, as indicated by Raman scattering on surface. The magnetic measurements showed the nanomaterial Fe3O4@Au@Cu is still magnetic although the gold shell and the functionalization with the copper complex have diminished the magnetization due to the dilution of the magnetic core. The nuclease assays performed with Fe3O4@Au@Cu indicate that the nuclease activity of the nanomaterial toward the plasmid DNA involves an oxidative pathway in which H2O2 species is involved as intermediate in a Fenton-like reaction. Based on the electron paramagnetic resonance spectra (aN = 15.07 G, aH = 14.99 G), such nuclease activity is assigned, essentially, to the HO species indicating that the radical production property of [Cu(2CP-Bz-SMe)]2+ is successfully transferred to the core-shell gold-coated Fe3O4 magnetic nanoparticles. To the best of our knowledge, this is the first study reporting nuclease activity due to the reactive oxygen species generated by a copper complex immobilized on a gold-coated magnetic nanoparticle.

Antimicrobial resistance and potential virulence of Vibrio parahaemolyticus isolated from water and bivalve mollusks from Bahia, Brazil.

The aim of the present study was to verify the antimicrobial susceptibility profile and virulence factors of Vibrio parahaemolyticus isolated from water and bivalve mollusks. A high percentage of V. parahaemolyticus was isolated in natura, processed bivalves tissues, and surrounding water (75%, 20%, and 59%, respectively). The most potential virulence phenotype in V. parahaemolyticus isolates was amylase production (97%) followed by DNase (83%), phospholipase (70%), ß-hemolytic activity (57%). The tdh and trh genes were not detected. Besides, a high antimicrobial resistance was observed for ampicillin (97%), minimum inhibitory concentration [MIC] = 400 µg and cephalothin (93%, MIC ≤ 100 µg). The absence of expression of tdh and trh virulence genes excluded the toxigenic potential of V. parahaemolyticus isolates; however, the high prevalence of antimicrobial resistance among the environmental strains is a risk to human health.

O papel das nucleases no laboratório de biologia molecular: vilãs ou aliadas? / The role of nucleases in the molecular biology laboratory: villains or allies?

Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.

In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.