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A deeper understanding of the donkey’s reproductive strategy is essential to improve reproductive efficiency in this species. In the jenny’s uterus, sperm selection relies on the post-insemination endometrial reaction, characterized by the influx of polymorphonuclear neutrophils (PMNs), which form extracellular traps (NETs) that capture spermatozoa. Of these, some are liberated through the action of a deoxyribonuclease (DNase) present in the seminal plasma. This study aimed to develop an in vitro model to evaluate the effects of NETs and DNase action on donkey sperm viability and motility. For this purpose, donkey ejaculates were collected, PMNs were isolated from the jenny’s blood and free spermatozoa were selected using a Silica gel filtration (EquiPure®, EP, Nidacon International AB, Mölndal, Sweden) to form five experimental groups: control (semen), SPZ-PMN, SPZ-PMN-DNase, SPZ-PMN-EP, SPZ-PMN-DNase-EP. Sperm viability and motility were assessed in all experimental groups using eosin-nigrosin staining and CASA sperm analysis system, respectively, at 1, 2 and 3 h of incubation. Sperm viability remained stable in the groups containing PMN, while it significantly (p < 0.05) decreased in the control group, suggesting a protective effect of PMN on sperm viability. Regarding kinetic parameters, spermatozoa initially captured by NETs and later liberated by DNase exhibited higher velocity (VCL, VSL, VAP), linearity (LIN), straightness (STR) and oscillation (WOB). These changes in sperm motility may be related to the physiological changes occurring before sperm capacitation. This study can improve the understanding of the unique reproductive strategy of donkeys and could be useful to improve in vitro sperm capacitation and embryo production.

Deoxyribonuclease I (DNase I) is an enzyme that hydrolyzes the phosphodiester bonds in the DNA backbone, enabling efficient DNA degradation. This activity is particularly relevant for degrading extracellular DNA (eDNA), a key structural component of the biofilm extracellular matrix that enhances bacterial attachment to implant surfaces and promotes cell-to-cell adhesion. By disrupting this matrix, DNase I offers significant potential to indirectly inhibit biofilm formation and reduce the risk of implant-associated infections (IAIs). In previous work, we developed a rapid electric field-assisted technique for producing anti-infective DNase I coatings on titanium (Ti) implant surfaces. To further evaluate clinical applicability, this study investigates the mechanical stability and in vitro cell compatibility of DNase I coatings applied to polydopamine (PDA)-functionalized Ti. Coatings were mechanically stressed using ultrasonication, followed by characterization of surface wettability and chemical composition. Compared to traditional dip-coating, AC-EPD-generated DNase I coatings exhibited greater stability, maintaining consistent wettability after 2 h of ultrasonication. Surface chemistry was examined using time-of-flight secondary ion mass spectrometry (ToF-SIMS), which detected amino acid fragments on both coating types. Notably, AC-EPD coatings contained a higher disulfide bond content, suggesting enhanced structural integrity. Furthermore, given the relevance to dental implant applications, human oral keratinocytes (HOKs) were used to assess cytotoxicity, cell adhesion, and spreading. The results indicated no cytotoxic effects, while promoting improved cell adhesion at both 24-h and 48-h incubation periods. Overall, these findings demonstrate that AC-EPD DNase I coatings are mechanically robust and biocompatible, making them a promising strategy for preventing implant-associated infections (IAIs) in dental implant applications.

Background: The role of cell-free DNA (cfDNA) in the pathogenesis of inflammatory bowel disease (IBD) has been recently suggested. The aim of this study was to analyze circulating cfDNA and deoxyribonuclease (DNase) activity in IBD patients in clinical remission. Materials and Methods: Plasma and serum were obtained from 72 patients with Crohn's disease and 28 patients with ulcerative colitis. Total cfDNA, nuclear DNA (ncDNA), mitochondrial DNA (mtDNA) and DNase activity were measured. Results: IBD patients showed higher levels of both ncDNA and mtDNA compared to healthy controls. Concentration of ncDNA was higher in males compared to females, including patients and healthy controls. However, unlike males higher amount of ncDNA was found in female IBD patients compared to healthy controls. DNase activity was significantly lower in male IBD patients compared with healthy controls. In addition, there was a negative correlation between DNase activity and ncDNA levels in male IBD patients. Conclusions: Herein we present increased amount of circulating ncDNA and mtDNA in IBD patients in clinical remission. Thus, unlike total cfDNA, circulating ncDNA and mtDNA might not represent the optimal biomarkers of disease activity. This is also the first report on sex difference in circulating ncDNA levels, possibly associated with lower DNase activity in males.

The HNH endonuclease superfamily usually contains a conserved HNH motif in the sequence, and the second subfamily of it uses N to replace the second H in the HNH motif. A bacterium with extracellular thermostable DNase was isolated and identified as Exiguobacterium sp. yc3. A 20 kDa putative DNase was later purified and the encoding gene of it was amplified and sequenced, the deduced amino acid sequence analysis showed that the protein belongs to the HNH endonuclease superfamily, and therefore it was named as EheA (ExiguobacteriumHNH Endonuclease). Characterization of the recombinant EheA confirmed that EheA is a DNase. By site-directed mutation method, H116, N141 and N156 were proved to be essential for the DNase activity. EheA is the first experimentally determined bacterial source endonuclease belonging to the second subfamily of HNH superfamily. Further bioinformatic analysis showed that EheA homologue genes are conserved in the Exiguobacterium species, which suggests their possible important functions for Exiguobacterium species. And as a thermostable DNase, EheA also has a promising future in many application fields. EheA is a novel bacterial source thermostable HNH superfamily endonuclease belonging to the second subfamily.

Deterministic lateral displacement (DLD) based microfluidic devices have been developed for capturing circulating tumor cells (CTCs) from the peripheral blood. There was frequent and problematic channel clogging around the micro-post array formed on a microchannel of the device. In this study, various agents were dispersed into the blood specimen to avoid clogging. At first, platelet aggregation was considered to be the cause of the clogging, but even plasmin, which was assumed to decompose platelet aggregations, did not show obvious inhibition of the clogging. Then, enzymes used for cell detachment from tissue were examined and decomposition of the clogging residue was observed. Finally, dispersion of deoxyribonuclease into a blood specimen was found to be effective for the inhibition of clogging. The existence of DNA in the clogging residue was also confirmed by propidium iodide (PI) staining, suggesting DNA adhering to the micro-post.

Study objectives: Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions. Design: Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation. Methods: Samples were separated into sol and gel (“mucous pellet”) phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 μmol/L. Results: HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase. Conclusions: The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.

Abstract The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.

Acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection is associated with high mortality rates and respiratory compromise in which excessive neutrophil extracellular trap (NET) production may amplify alveolar inflammation and injury. Dornase alfa, a recombinant DNAse 1, has been proposed to attenuate these effects by degrading extracellular DNA and enhancing alveolar clearance of NETs. In this multicenter, open-label, randomized in two parallel arms (1:1) controlled trial, intubated COVID-19 ARDS patients received either standard-of-care (SOC) alone or SOC plus aerosolized dornase alfa (2500 IU twice daily for 7 days). The primary endpoint was the proportion of patients with ARDS severity improvement at Day 7, defined by at least one-grade improvement on the Berlin criteria scale. Secondary outcomes included 28-day mortality, ventilator-free days, ICU-free days, and changes in key ventilatory parameters. Biological samples were analyzed to assess NET related markers, DNAse drug activity and indicate possible bioavailability issues associated with aerosolization of dornase alfa. Seventy-seven patients were enrolled (dornase alfa group, n  = 39; SOC group, n  = 38). At Day 7, ARDS severity improved in 18% of patients receiving dornase alfa compared with 29% in the SOC group (adjusted OR: 0.33; 95% CI 0.09–1.14; p  = 0.11). Secondary endpoints, including 28-day mortality, ventilator-free days, and ICU-free days, showed no significant differences between groups. Adverse events occurred in 38.5% of patients in the dornase alfa arm versus 31.6% in the SOC arm, indicating comparable safety profiles. Despite early increases in NET plasmatic levels observed in both groups and successful ex vivo NET degradation, aerosolized dornase alfa failed to significantly enhance DNAse activity or reduce NET-related markers in patients’ plasma and mucus, suggesting potential bioavailability limitations with this delivery method. In patients with COVID-19-related ARDS, dornase alfa did neither significantly reduce ARDS severity nor improve clinical outcomes over SOC. Although well tolerated, analysis of biological samples suggests that aerosol administration may have compromised drug bioavailability. Further trials are needed to determine whether specific patient subgroups could benefit more from dornase alfa or if alternative drug delivery methods might enhance treatment efficacy. ClinicalTrials.gov, NCT04355364. Registered on 21/04/2020.

Persistent or refractory apical periodontitis is primarily caused by microbial retention, as conventional root canal treatment often fails to eliminate infections completely, and systemic antibiotic therapy is insufficient to achieve effective concentrations for eradicating bacterial biofilms within root canals. This highlights the urgent need for novel therapeutics offering safe and effective antimicrobial strategies. Antimicrobial photodynamic therapy (aPDT) is a promising approach for root canal disinfection. However, commonly used photosensitizers such as Ce6 suffer from poor water solubility and strong aggregation tendencies, resulting in limited penetration into infected sites. In this study, we developed a DNase I-Lip@Ce6 nanodelivery system by combining deoxyribonuclease I (DNase I) with liposome-encapsulated Ce6. The liposomal carrier facilitated efficient delivery of Ce6 into target bacterial cells, while DNase I degraded extracellular DNA in the biofilm matrix, weakening its protective barrier. This synergistically enhanced Ce6 penetration and therapeutic efficacy, leading to the successful eradication of planktonic Enterococcus faecalis and in vitro biofilms. This strategy offers a novel approach for the precision treatment of persistent oral infections and holds strong potential for clinical translation.