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Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In a recent issue of Cell Reports, Maurya et al. 1 demonstrated that Drep4, the Drosophila homolog of caspase-activated DNase (CAD), drives DNA strand breaks during myeloid-like/macrophage cell differentiation and that this Drep4/CAD activity is essential for the differentiation process., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogenous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

DNA is the genetic code found inside all living cells and its molecular stability can also be utilized outside the cell. While extracellular DNA (eDNA) has been identified as a structural polymer in bacterial biofilms, whether it persists stably throughout development remains unclear. Here, we report that eDNA is temporarily invested in the biofilm matrix before being reclaimed later in development. Specifically, by imaging eDNA dynamics within undomesticated Bacillus subtilis biofilms, we found eDNA is produced during biofilm establishment before being globally degraded in a spatiotemporally coordinated pulse. We identified YhcR, a secreted Ca 2+ -dependent nuclease, as responsible for eDNA degradation in pellicle biofilms. YhcR cooperates with two other nucleases, NucA and NucB, to reclaim eDNA for its phosphate content in colony biofilms. Our results identify extracellular nucleases that are crucial for eDNA reclamation during biofilm development and we therefore propose a new role for eDNA as a dynamic metabolic reservoir., (© 2024. The Author(s).)

Bacterial biofilm-related infections have become a significant global concern in public health and economy. Extracellular DNA (eDNA) is regarded as one of the key elements of extracellular polymeric substances (EPS) in bacterial biofilm, providing robust support to maintain the stability of bacterial biofilms for fighting against environmental stresses (such as antibiotics, reactive oxygen species (ROS), and hyperthermia). In this study, ternary AuAgCu hydrogels nanozyme with porous network structures were utilized for the immobilization of DNase (AuAgCu@DNase hydrogels) to realize enhanced biofilm decomposition and antibacterial therapy of MRSA. The prepared AuAgCu@DNase hydrogels can efficiently hydrolyze eDNA in biofilms so that the generated ROS and hyperthermia by laser irradiation can permeate into the interior of the biofilm to achieve deep sterilization. The typical interface interactions between AuAgCu hydrogels and DNase and the excellent photothermal-boost peroxidase-like performances of AuAgCu hydrogels take responsibility for the enhanced antibacterial activity. In the MRSA-infected wounds model, the in vivo antibacterial results revealed that the AuAgCu@DNase hydrogels possess excellent drug-resistant bacteria-killing performance with superb biocompatibility. Meanwhile, the pathological analysis of collagen deposition and fibroblast proliferation of wounds demonstrate highly satisfactory wound healing. This work offers an innovative path for developing nanozyme-enzyme antibacterial composites against drug-resistant bacteria and their biofilms., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies 1 . Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events 1,2 . However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events., (© 2024. The Author(s).)

Planktonic bacteria tend to form sessile community architectures to shield resident bacteria from various environmental stresses. The formed biofilm leads to the failure of conventional antimicrobial therapy. Extracellular macromolecules, including extracellular DNA (eDNA), proteins, lipids, and polysaccharides, crosslink into gel-like structures through electrostatic forces in the mature biofilm matrix. The stereo-structural integrity and chemical inertia of the extracellular polymeric matrix result in comprehensive antimicrobial resistance to antibacterial polysaccharides. Herein, an ionic gelation method was employed to functionalize cationic chitosan nanoparticles (CSNPs) with chloroquine and deoxyribonuclease. The modification involved shifting eDNA chirality through a DNA-intercalating agent, chloroquine, and hydrolyzing an eDNA scaffold with deoxyribonuclease. The antibiofilm activity was assessed against a standard Staphylococcus aureus strain and clinical subtype isolates. Functional modifications targeting eDNA improved the chitosan anti-biofilm efficiency (residual biomass decreased from 74.2 to 90.3 % to 16.7-24.6 %) by disrupting the biofilm matrix. The functional CSNPs worked as a sensitizer prodrug, contributing to a bactericidal process of chitosan itself (cell wall damage increased from 11.38-18.16 % to 55.2-61.4 %) by dispersing the biofilm-enclosed bacteria. In vivo, the bacterial burden of infected mouse joints was reduced by 4.1 lg CFU/mL. Our results indicate the potential of this chitosan-based anti-infection strategy in biofilm-related infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cellular senescence is a response to many stressful insults. DNA damage is a consistent feature of senescent cells, but in many cases its source remains unknown. Here, we identify the cellular endonuclease caspase-activated DNase (CAD) as a critical factor in the initiation of senescence. During apoptosis, CAD is activated by caspases and cleaves the genomic DNA of the dying cell. The CAD DNase is also activated by sub-lethal signals in the apoptotic pathway, causing DNA damage in the absence of cell death. We show that sub-lethal signals in the mitochondrial apoptotic pathway induce CAD-dependent senescence. Inducers of cellular senescence, such as oncogenic RAS, type-I interferon, and doxorubicin treatment, also depend on CAD presence for senescence induction. By directly activating CAD experimentally, we demonstrate that its activity is sufficient to induce senescence in human cells. We further investigate the contribution of CAD to senescence in vivo and find substantially reduced signs of senescence in organs of ageing CAD-deficient mice. Our results show that CAD-induced DNA damage in response to various stimuli is an essential contributor to cellular senescence., (© 2024. The Author(s).)

The coral reef microbiome plays a vital role in the health and resilience of reefs. Previous studies have examined phage therapy for coral pathogens and for modifying the coral reef microbiome, but defence systems against coral-associated bacteria have received limited attention. Phage defence systems play a crucial role in helping bacteria fight phage infections. In this study, we characterized a new defence system, Hma (HmaA-HmaB-HmaC), in the coral-associated Halomonas meridiana derived from the scleractinian coral Galaxea fascicularis. The Swi2/Snf2 helicase HmaA with a C-terminal nuclease domain exhibits antiviral activity against Escherichia phage T4. Mutation analysis revealed the nickase activity of the nuclease domain (belonging to PDD/EXK superfamily) of HmaA is essential in phage defence. Additionally, HmaA homologues are present in ~1000 bacterial and archaeal genomes. The high frequency of HmaA helicase in Halomonas strains indicates the widespread presence of these phage defence systems, while the insertion of defence genes in the hma region confirms the existence of a defence gene insertion hotspot. These findings offer insights into the diversity of phage defence systems in coral-associated bacteria and these diverse defence systems can be further applied into designing probiotics with high-phage resistance., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection., (© 2024. The Author(s).)

Type VI secretion system (T6SS) is a potent weapon employed by various Pseudomonas species to compete with neighboring microorganisms for limited nutrients and ecological niches. However, the involvement of T6SS effectors in interbacterial competition within the phytopathogen Pseudomonas syringae remains unknown. In this study, we examined two T6SS clusters in a wild-type P. syringae MB03 and verified the involvement of one cluster, namely, T6SS-1, in interbacterial competition. Additionally, our results showed that two T6SS DNase effectors, specifically Tde1 and Tde4, effectively outcompeted antagonistic bacteria, with Tde4 playing a prominent role. Furthermore, we found several cognate immunity proteins, including Tde1i a , Tde1i b , and Tde4i, which are located in the downstream loci of their corresponding effector protein genes and worked synergistically to protect MB03 cells from self-intoxication. Moreover, expression of either Tde1 or C-terminus of Tde4 in Escherichia coli cells induced DNA degradation and changes in cell morphology. Thus, our results provide new insights into the role of the T6SS effectors of P. syringae in the interbacterial competition in the natural environment., Importance: The phytopathogen Pseudomonas syringae employs an active type VI secretion system (T6SS) to outcompete other microorganisms in the natural environment, particularly during the epiphytic growth in the phyllosphere. By examining two T6SS clusters in P. syringae MB03, T6SS-1 is found to be effective in killing Escherichia coli cells. We highlight the excellent antibacterial effect of two T6SS DNase effectors, namely, Tde1 and Tde4. Both of them function as nuclease effectors, leading to DNA degradation and cell filamentation in prey cells, ultimately resulting in cell death. Our findings deepen our understanding of the T6SS effector repertoires used in P. syringae and will facilitate the development of effective antibacterial strategies., Competing Interests: The authors declare no conflict of interest.

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes , providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri . To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451 , showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae . However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437 , a homolog of V. cholerae dns , might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes . We find that nuclease activity is an important determinant of colonization in V. fischeri , broadening our understanding of how microbes establish and maintain beneficial associations., Competing Interests: The authors declare no conflict of interest.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7 DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7 DjTatD can be considered highly effective antibiofilm agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 ( n = 439) and from healthy controls ( n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation. NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation 1 . Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET) 2 . Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Enteroaggregative E. coli (EAEC) is a major cause of diarrhea worldwide. EAEC are highly adherent to cultured epithelial cells and make biofilms. Both adherence and biofilm formation rely on the presence of aggregative adherence fimbriae (AAF). We compared biofilm formation from two EAEC strains of each of the five AAF types. We found that AAF type did not correlate with the level of biofilm produced. Because the composition of the EAEC biofilm has not been fully described, we stained EAEC biofilms to determine if they contained protein, carbohydrate glycoproteins, and/or eDNA and found that EAEC biofilms contained all three extracellular components. Next, we assessed the changes to the growing or mature EAEC biofilm mediated by treatment with proteinase K, DNase, or a carbohydrate cleavage agent to target the different components of the matrix. Growing biofilms treated with proteinase K had decreased biofilm staining for more than half of the strains tested. In contrast, although sodium metaperiodate only altered the biofilm in a quantitative way for two strains, images of biofilms treated with sodium metaperiodate showed that the EAEC were more spread out. Overall, we found variability in the response of the EAEC strains to the treatments, with no one treatment producing a biofilm change for all strains. Finally, once formed, mature EAEC biofilms were more resistant to treatment than biofilms grown in the presence of those same treatments., Competing Interests: Author VV was employed by Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Van Nederveen and Melton-Celsa.)

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry., (© 2024 Wiley-VCH GmbH.)

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Irritable bowel syndrome (IBS) involves low-grade mucosal inflammation. Among the various approaches capable of managing the symptoms, physical activity is still under investigation. Despite its benefits, it promotes oxidative stress and inflammation. Mitochondria impacts gut disorders by releasing damage-associated molecular patterns, such as cell-free mtDNA (cf-mtDNA), which support inflammation. This study evaluated the effects of a 12-week walking program on the cf-mtDNA and DNase in 26 IBS and 17 non-IBS subjects. Pro- and anti-inflammatory cytokines were evaluated by ELISA. Digital droplet PCR was used to quantify cf-mtDNA; DNase activity was assessed using a single radial enzyme diffusion assay. PCR-RFLP was used to genotype DNASE1 rs1053874 SNP. Significantly lower IL-10 levels were found in IBS than in non-IBS individuals. Exercise reduced cf-mtDNA in non-IBS subjects but not in IBS patients. DNase activity did not correlate with the cf-mtDNA levels in IBS patients post-exercise, indicating imbalanced cf-mtDNA clearance. Different rs1053874 SNP frequencies were not found between groups. The study confirms the positive effects of regular moderate-intensity physical activity in healthy subjects and its role in cf-mtDNA release and clearance. Walking alone might not sufficiently reduce subclinical inflammation in IBS, based on imbalanced pro- and anti-inflammatory molecules. Prolonged programs are necessary to investigate their effects on inflammatory markers in IBS.

Aims: Neutrophil extracellular trap (NET), which is formed by DNA threads, induces septic shock by aggravating systemic inflammation. An intravenous administration of deoxyribonuclease is regarded as a compelling modality for treating septic shock. However, alternative routes should be chosen when cutaneous veins are all collapsed due to hypotension. In this study, we genetically engineered this enzyme to develop a rectal suppository formulation to treat septic shock., Main Methods: Dnase1 was mutated at two amino acid residues to increase its stability in the blood and fused with a cell-penetrating peptide CR8 to increase its absorption through the rectal mucosa, which is designated AR-CR8. The life-saving effect of AR-CR8 was evaluated in a LPS-induced shock mouse model., Key Findings: AR-CR8 was shown to remove NETs effectively in human neutrophils. When AR-CR8 was administered to the mouse rectum, the deoxyribonuclease activity in the mouse serum was significantly increased. In the LPS-induced shock model, 90 % of the control mice died over 72 h after LPS injection. In contrast, the rectal administration of AR-CR8 showed a mortality rate of 30 % by 72 h after LPS injection. The Log-rank test revealed that the survival rate is significantly higher in the AR-CR8 group. The NET markers in the mouse serum were enhanced by LPS, and significantly downregulated in the AR-CR8 group. These results suggest that AR-CR8 ameliorates LPS-induced shock by degrading NETs., Significance: The engineered DNASE1 could be developed as a rectal suppository formulation to treat septic shock urgently at out-of-hospital places where no syringe is available., Competing Interests: Declaration of competing interest The authors have no potential conflicts., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration., Methods: Six different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR)., Results: Out of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR., Conclusion: The HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy., (© 2024. The Author(s).)

The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont Wolbachia, alter long noncoding RNA (lncRNA) and DNA during Drosophila sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.

Background: Thrombo-inflammation and neutrophil extracellular traps (NETs) are exacerbated in severe cases of COVID-19, potentially contributing to disease exacerbation. However, the mechanisms underpinning this dysregulation remain elusive. We hypothesised that lower DNase activity may be associated with higher NETosis and clinical worsening in patients with COVID-19., Methods: Biological samples were obtained from hospitalized patients (15 severe, 37 critical at sampling) and 93 non-severe ambulatory cases. Our aims were to compare NET biomarkers, functional DNase levels, and explore mechanisms driving any imbalance concerning disease severity., Results: Functional DNase levels were diminished in the most severe patients, paralleling an imbalance between NET markers and DNase activity. DNase1 antigen levels were higher in ambulatory cases but lower in severe patients. DNase1L3 antigen levels remained consistent across subgroups, not rising alongside NET markers. DNASE1 polymorphisms correlated with reduced DNase1 antigen levels. Moreover, a quantitative deficiency in plasmacytoid dendritic cells (pDCs), which primarily express DNase1L3, was observed in critical patients. Analysis of public single-cell RNAseq data revealed reduced DNase1L3 expression in pDCs from severe COVID-19 patient., Conclusion: Severe and critical COVID-19 cases exhibited an imbalance between NET and DNase functional activity and quantity. Early identification of NETosis imbalance could guide targeted therapies against thrombo-inflammation in COVID-19-related sepsis, such as DNase administration, to avert clinical deterioration., Trial Registration: COVERAGE trial (NCT04356495) and COLCOV19-BX study (NCT04332016)., (© 2024. The Author(s).)

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

Deoxyribonuclease 1-like 3 (DNASE1L3) has been shown to play nonnegligible roles in several types of carcinomas. Nevertheless, the biological function, clinical relevance, and influence of DNASE1L3 in colorectal cancer (CRC) remain obscure. Immunohistochemistry was adopted to examine DNASE1L3 and CDKN1A expression in CRC tissue, and the clinical significance of DNASE1L3 was assessed. Cell counting kit-8, colony formation, and transwell assays were employed for assessing tumor proliferation and migration. The mechanisms underlying the impact of DNASE1L3 were explored via western blot analysis, co-immunoprecipitation, and ubiquitination assay. It was observed that DNASE1L3 was downregulated in CRC tissues and was tightly associated with patient prognosis. DNASE1L3 impaired CRC cell proliferation and migration through elevating CDKN1A via suppressing CDKN1A ubiquitination. Meanwhile, DNASE1L3 was positively related to CDKN1A. In mechanism, DNASE1L3 and CDKN1A interacted with the E3 ubiquitin ligase NEDD4. Moreover, DNASE1L3 was competitively bound to NEDD4, thus repressing NEDD4-mediated CDKN1A ubiquitination and degradation. These discoveries implied the potential mechanisms of DNASE1L3 during tumorigenesis, suggesting that DNASE1L3 may serve as a new potential therapeutic agent for CRC., (© 2023 International Federation for Cell Biology.)

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 (Padi4 flox/flox LysM CRE ). 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., (© 2023 British Pharmacological Society.)

Reinforced biofilm structures and dysfunctional neutrophils induced by excessive oxidative stress contribute to the refractoriness of diabetes-related biofilm infections (DRBIs). Herein, in contrast to traditional antibacterial therapies, an immune switchpoint-driven neutrophil immune function conversion strategy based on a deoxyribonuclease I loaded vanadium carbide MXene (DNase-I@V 2 C) nanoregulator is proposed to treat DRBIs via biofilm lysis and redirecting neutrophil functions from NETosis to phagocytosis in diabetes. Owing to its intrinsic superoxide dismutase/catalase-like activities, DNase-I@V 2 C effectively scavenges reactive oxygen species (ROS) in a high oxidative stress microenvironment to maintain the biological activity of DNase-I. By increasing the depth of biofilm penetration of DNase-I, DNase-I@V 2 C thoroughly degrades extracellular DNA and neutrophil extracellular traps (NETs) in extracellular polymeric substances, thus breaking the physical barrier of biofilms. More importantly, as an immune switchpoint regulator, DNase-I@V 2 C can skew neutrophil functions from NETosis toward phagocytosis by intercepting ROS-NE/MPO-PAD4 and activating ROS-PI3K-AKT-mTOR pathways in diabetic microenvironment, thereby eliminating biofilm infections. Biofilm lysis and synergistic neutrophil function conversion exert favorable therapeutic effects on biofilm infections in vitro and in vivo. This study serves as a proof-of-principle demonstration of effectively achieving DRBIs with high therapeutic efficacy by regulating immune switchpoint to reverse neutrophil functions., (© 2023 Wiley-VCH GmbH.)

We describe a new way to trigger mRNA degradation in Saccharomyces cerevisiae synthetic gene circuits. Our method demands to modify either the 5'- or the 3'-UTR that flanks a target gene with elements from the pre-crRNA of type V Cas12a proteins and expresses a DNase-deficient Cas12a (dCas12a). dCas12a recognizes and cleaves the pre-crRNA motifs on mRNA sequences. Our tool does not require complex engineering operations and permits an efficient control of protein expression via mRNA degradation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We have developed a novel method for genomic footprinting of transcription factors (TFs) that detects potential gene regulatory relationships from DNase-seq data at the nucleotide level. We introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. A mild cross-linking step in XL-DNase-seq improves the detection of DNase-based footprints of dynamic TFs. The footprint strengths and detectability depend on an optimal cross-linking procedure. This method may help extract novel gene regulatory circuits involving previously undetectable TFs. The XL-DNase-seq method is illustrated here for activated mouse macrophage-like cells, which share several features with inflammatory macrophages., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation 1-5 . Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes 1,6,7 , but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion., (© 2023. The Author(s).)

Background: Hepatic ischemia-reperfusion injury (IRI) is a common innate immune-mediated sterile inflammatory response in liver transplantation and liver tumor resection. Neutrophil extracellular traps (NETs) can aggravate liver injury and activates innate immune response in the process of liver IRI. However, Curcumin (Cur) can reverse this damage and reduce NETs formation. Nevertheless, the specific regulatory mechanism is still unclear in liver IRI. This study aimed to explore the potential mechanisms that how does Cur alleviate hepatic IRI by inhibits NETs production and develop novel treatment regimens., Methods: We established a hepatic IRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 2 h, 6 h, 12 h, and 24 h respectively. Subsequently, we were separated into 5 groups, namely the I/R group, Cur group, DNase-1 group, Cur + DNase1 group and sham operation group. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST), Hematoxylin-eosin staining, immunofluorescence, and TUNEL analysis were applied to assess liver injury degree and NETs levels. Western blot assay was used to detect the protein levels of apoptosis-related proteins and MEK pathway proteins., Results: Cur could alleviate hepatic IRI by inhibiting the generation of NETs via suppressing the MEK/ERK pathway. In addition, this study also revealed that DNase-1 is vital for alleviating hepatic IRI by reducing the generation of NETs., Conclusions: Cur combined with DNase-1 was more effective than the two drugs administered alone in alleviating hepatic IRI by inhibiting the generation of NETs. These results also suggested that curcumin combined with DNase-1 was a potential therapeutic strategy to mitigate hepatic IRI.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs' architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis-associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell-free mitochondrial DNA (cf-mtDNA) in the regulation of alveolar macrophage activation during sepsis-associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase-I) and human serum albumin (HSA) via glutaraldehyde-mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf-mtDNA clearance. The synthesized DNase-I/HSA NMs are endowed with self-propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase-I/HSA NMs effectively eliminates cf-mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf-mtDNA clearance strategy using DNase-I/HSA NMs is considered to be an attractive approach for sepsis-associated ALI., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The analysis and improvement of DNA nanostructure biostability is one of the keys areas of progress needed in DNA nanotechnology applications. Here, we present a plate-compatible fluorometric assay for measuring DNA nanostructure biostability using the common intercalator ethidium bromide. We demonstrate the assay by testing the biostability of duplex DNA, a double crossover DNA motif, and a DNA origami nanostructure against different nucleases and in fetal bovine serum. This method scales well to measure a large number of samples using a plate reader and can complement existing methods for assessing and developing robust DNA nanostructures.

Pioneer transcription factors interact with nucleosomes to scan silent, compact chromatin, enabling cooperative events that modulate gene activity. While at a subset of sites pioneer factors access chromatin by assisted loading with other transcription factors, the nucleosome-binding properties of pioneer factors enable them to initiate zygotic genome activation, embryonic development, and cellular reprogramming. To better understand nucleosome targeting in vivo, we assess whether pioneer factors FoxA1 and Sox2 target stable or unstable nucleosomes and find that they target DNase-resistant, stable nucleosomes, whereas HNF4A, a non-nucleosome binding factor, targets open, DNase-sensitive chromatin. Despite FOXA1 and SOX2 targeting similar proportions of DNase-resistant chromatin, using single-molecule tracking, we find that FOXA1 uses lower nucleoplasmic diffusion and longer residence times while SOX2 uses higher nucleoplasmic diffusion and shorter residence times to scan compact chromatin, while HNF4 scans compact chromatin much less efficiently. Thus, pioneer factors target compact chromatin through distinct processes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon modification. USCs from a healthy donor sample were isolated according to established protocols. Within 2 months, 10 clones had been expanded, analyzed, and frozen. Fluorescence-activated cell sorting analysis of individual clones revealed that all 10 clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90, and CD146; negative for CD31, CD34, and CD45). The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN (NPHS1), podocalyxin, and Wilms tumor 1 (WT1). Transfection of USCs with a strongly expressing Green fluorescent protein plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by live animal imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, USCs represent a clinically relevant cell type that can express nonviral transgenes. Impact statement Acute kidney injury (AKI) affects over 13 million people worldwide each year, with hospitalization rates on the rise. There are no therapies that directly regenerate the kidney after AKI. Each human kidney contains approximately one million nephrons that process ∼100 L of urinary filtrate each day. Thousands of kidney cells become detached and are excreted in the urine. A small percentage of these cells can be clonally derived into urine-derived stem cells. We have optimized methods for genome engineering of adult human urine-derived stem cells for future applications in regenerative approaches to treat kidney injury.

Scope: Mitochondrial DNA (mtDNA) released into the cytosol serves as a member of damage-associated molecular patterns to initiate inflammatory responses. Mangiferin is a xanthonoid derivative, usually isolated from plants including mangoes and iris unguicularis. This study aims to investigate whether mangiferin prevents mtDNA accumulation in the cytosol with a focus on deoxyribonuclease 2 (DNase 2) protection from oxidative damage., Methods and Results: Mangiferin administration effectively protects against hepatotoxicity in mice subjected to CCl 4 challenge or bile duct ligation (BDL) surgery. Moreover, mangiferin activates nuclear factor erythroid 2-related factor (Nrf2)-antioxidant signaling, reduces cytosolic mtDNA accumulation, and suppresses Toll-like receptor 9 (TLR-9)/myeloid differentiation factor 88 (MyD88)-dependent inflammation in the liver. The study prepares hepatic mtDNA to stimulate hepatocytes, and finds that mangiferin protects DNase 2 protein abundance. mtDNA induces reactive oxygen species (ROS) production to promote DNase 2 protein degradation through oxidative modification, but mangiferin protects DNase 2 protein stability in a Nrf2-dependent manner. In hepatic Nrf2 deficiency mice, the study further confirms that Nrf2 induction is required for mangiferin to clear cytosolic mtDNA and block mtDNA-mediated TLR9/MyD88/nuclear factor kappa-B (NF-κB) inflammatory signaling cascades., Conclusion: These findings provide new insights into the role of mangiferin as a liver protecting agent, and suggest protection of DNase 2 as a novel therapeutic strategy for pharmacological intervention to prevent liver damage., (© 2023 Wiley-VCH GmbH.)

Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents., (© 2023 Lacey et al.)

Prokaryotic Argonaute (pAgo) proteins are programmable nucleases with great promise in genetic engineering and biotechnology. Previous studies identified several DNA-targeting pAgo nucleases from mesophilic and thermophilic prokaryotic species that are active in various temperature ranges. However, the effects of temperature on the specificity of target recognition and cleavage by pAgos have not been studied. Here, we describe a thermostable pAgo nuclease from the thermophilic bacterium Thermobrachium celere, TceAgo. We show that TceAgo preferentially uses 5'-phosphorylated small DNA guides and can perform specific cleavage of both single-stranded and double-stranded DNA substrates in a wide range of temperatures. Single-nucleotide mismatches between guide and target molecules differently change the reaction efficiency depending on the mismatch position, with the fidelity of target recognition greatly increased at elevated temperatures. Thus, TceAgo can serve as a tool to allow specific detection and cleavage of DNA targets in a temperature-dependent manner. The results demonstrate that the specificity of programmable nucleases can be strongly affected by the reaction conditions, which should be taken into account when using these nucleases in various in vitro and in vivo applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca 2+ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca 2+ is involved in the regulation of cellular Ca 2+ homeostasis. However, it remains unclear whether nuclear Ca 2+ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca 2+ levels are associated with nuclear apoptosis. In this study, changes in Ca 2+ levels, Ca 2+ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca 2+ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca 2+ levels ([Ca 2+ ] NE ) and nucleocytoplasmic Ca 2+ levels ([Ca 2+ ] NC ) increased by 78% ( p < 0.01) and 106% ( p < 0.01), respectively. The levels of Ca 2+ -ATPase type 2 (Ca 2+ -ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP 3 R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP 3 ) increased by 470% ( p < 0.001), 94% ( p < 0.05), 170% ( p < 0.001), 640% ( p < 0.001) and 12% ( p < 0.05), respectively, and the levels of Na + /Ca 2+ exchanger 3 (NCX3), Ca 2+ /calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% ( p < 0.001), 33% ( p < 0.05) and 5% ( p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca 2+ uptake from cytoplasm is involved in the increase in [Ca 2+ ] NE after hindlimb unloading. Moreover, the increase in [Ca 2+ ] NC is attributed to increased Ca 2+ release into nucleocytoplasm and weakened Ca 2+ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca 2+ ] NC , leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca 2+ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle.

During reactivation from latency, gammaherpesviruses radically restructure their host cell to produce virion particles. To achieve this and thwart cellular defenses, they induce rapid degradation of cytoplasmic mRNAs, suppressing host gene expression. In this article, we review mechanisms of shutoff by Epstein-Barr virus (EBV) and other gammaherpesviruses. In EBV, canonical host shutoff is accomplished through the action of the versatile BGLF5 nuclease expressed during lytic reactivation. We explore how BGLF5 induces mRNA degradation, the mechanisms by which specificity is achieved, and the consequences for host gene expression. We also consider non-canonical mechanisms of EBV-induced host shutoff. Finally, we summarize the limitations and barriers to accurate measurements of the EBV host shutoff phenomenon.

Cell nucleus is the desired subcellular organelle of many therapeutic drugs. Although numerous nanomaterial-based methods have been developed which could facilitate nuclear-targeted delivery of small-molecule drugs, few are known to be capable of delivering exogenous native proteins. Herein, we report a convenient and highly robust approach for effective nuclear-targeted delivery of native proteins/antibodies by using biodegradable silica nanocapsules (BSNPs) that were surface-modified with different nuclear localization signals (NLS) peptides. We found that, upon gaining entry to mammalian cells via endocytosis, such nanocapsules (protein@BSNP-NLS) could effectively escape from endolysosomal vesicles with the assistance of an endosomolytic peptide (i.e., L17E), accumulate in cell nuclei and release the encapsulated protein cargo with biological activities. Cloaked with HeLa cell membrane, DNase@BSNP-NLS/L17E-M (with L17E encapsulated) homologously delivered functional proteins to cancer cell nuclei in tumor-xenografted mice. In vitro and in vivo anti-tumor properties, such as long blood circulation time and effective tumor growth inhibition, indicate that the nuclear-targeted cell-membrane-cloaked BSNPs (DNase@BSNP-NLS/L17E-M) platform is a promising therapeutic approach to nuclear related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Many Gram-negative bacteria deploy a type VI secretion system (T6SS) to inject toxins into target cells to promote their survival and replication in complex environments. Here, we report that Acinetobacter baumannii uses its T6SS to kill fungi and that the effector TafE ( ACX60&#95;15365 ) is responsible for such killing. Although ectopically expressed TafE is toxic to both Escherichia coli and Saccharomyces cerevisiae, deletion of tafE only affects the antifungal activity of A. baumannii. We demonstrate that TafE is a DNase capable of targeting the nuclei of yeast cells and that an Ntox15 domain is essential for its ability to degrade DNA. Furthermore, our findings show that A. baumannii is protected from the toxicity of TafE by elaborating the immunity protein TaeI ( ACX60&#95;15360 ), which antagonizes the activity of the effector by direct binding. The discovery of A. baumannii T6SS effectors capable of killing multiple taxonomically distinct microbes has shed light on a mechanism of the high-level fitness of this pathogen in environments characterized by scarce nutrients and the potential presence of diverse microorganisms. IMPORTANCE Acinetobacter baumannii is an increasing important nosocomial pathogen that is difficult to combat due to its ability to survive in harsh environments and the emergence of isolates that are resistant to multiple antibiotics. A better understanding of the mechanism underlying the toughness of A. baumannii may identify its Achilles' heel, which will facilitate the development of novel preventive and treatment measures. In this study, our findings show that A. baumannii kills fungi with the DNase effector TafE injected into competitor cells by its type VI secretion system. A. baumannii is protected from the activity of TafE by the immunity protein TaeI, which inactivates the effector by direct binding. Our results suggest that inactivation of its T6SS or effectors may reduce the fitness of A. baumannii and increase the effectiveness of treatment by means such as antibiotics. Furthermore, our finding suggests that targeted degradation of TaeI may be an effective strategy to kill A. baumannii.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.

Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We previously reported evidence of an autoimmune signature in HS, characterized by enhanced neutrophil extracellular trap (NET) infiltration in HS skin lesions and dysregulation of the adaptive immune system characterized by the presence of autoantibodies. Timely removal of NETs is critical for tissue homeostasis to prevent a dysregulated generation of modified autoantigens and tissue damage. DNases 1 and 1L3 play important roles in proper NET removal. We tested the hypothesis that NETs in patients with HS are not effectively cleared owing to the presence of antibodies against DNase 1 and DNase 1L3. We report that HS serum poorly degraded NETs. Addition of exogenous DNase 1 restored NET degradation capabilities in a subset of HS samples. DNase 1 activity was significantly decreased in HS sera. Anti‒DNase 1 and ‒DNase 1L3 antibodies were detected in serum samples and skin lesions from patients with HS. Purified IgGs from HS decreased DNase 1 activity and NET degradation. Taken together, this identification of neutralizing antibodies against nucleases in HS expands the understanding of the pathogenesis of this disease to support an autoimmune mechanism in its underlying pathogenesis., (Published by Elsevier Inc.)

Background: Eukaryotic gene expression is controlled by cis-regulatory elements (CREs), including promoters and enhancers, which are bound by transcription factors (TFs). Differential expression of TFs and their binding affinity at putative CREs determine tissue- and developmental-specific transcriptional activity. Consolidating genomic datasets can offer further insights into the accessibility of CREs, TF activity, and, thus, gene regulation. However, the integration and analysis of multimodal datasets are hampered by considerable technical challenges. While methods for highlighting differential TF activity from combined chromatin state data (e.g., chromatin immunoprecipitation [ChIP], ATAC, or DNase sequencing) and RNA sequencing data exist, they do not offer convenient usability, have limited support for large-scale data processing, and provide only minimal functionality for visually interpreting results., Results: We developed TF-Prioritizer, an automated pipeline that prioritizes condition-specific TFs from multimodal data and generates an interactive web report. We demonstrated its potential by identifying known TFs along with their target genes, as well as previously unreported TFs active in lactating mouse mammary glands. Additionally, we studied a variety of ENCODE datasets for cell lines K562 and MCF-7, including 12 histone modification ChIP sequencing as well as ATAC and DNase sequencing datasets, where we observe and discuss assay-specific differences., Conclusion: TF-Prioritizer accepts ATAC, DNase, or ChIP sequencing and RNA sequencing data as input and identifies TFs with differential activity, thus offering an understanding of genome-wide gene regulation, potential pathogenesis, and therapeutic targets in biomedical research., (© The Author(s) 2023. Published by Oxford University Press GigaScience.)

Biomimetic mineralization of enzymes for enhanced stability and activity is an important area of research due to its potential applications. Inorganic materials with enzymes coated and or embedded in them, viz., protein-inorganic hybrid nanomaterials with distinctive morphology and surface characteristics are promising candidates for exploring their elevated enzymatic activity. In this work, we have developed two different types of protein inorganic nanohybrid materials using a 120 kDa lectin purified from bitter gourd seeds (Momordica charantia lectin, MCL), and (i) copper phosphate nanoflowers to result in a protein - inorganic nano hybrid material CuPNF&#95;MCL and (ii) encapsulating the protein in zeolitic imidazole framework, ZIF8&#95;MCL. While CuPNF&#95;MCL showed floral morphology, the ZIF8&#95;MCL mostly showed hexapod morphology as noticed from the microscopy data. Both the nanomaterials showed a distinctive trend of decrease in size with increase in the protein concentration used during the preparation. The nanoflowers also showed an increase in the tightness of the packing of petals with increase in the protein concentration. Powder X-Ray diffraction studies confirmed the crystallinity of the inorganic frameworks. The Fourier Transform infrared spectroscopy studies coupled with confocal imaging of the fluorophore tagged MCL embedded hybrids confirmed the presence of the protein. The MCL protein was examined for its ability to cleave DNA, i.e., nuclease activity using pBR322, wherein the form I plasmid is completely transformed into the form II / III at 2 mg/mL concentration of the protein. However, both the hybrids showed a superior nuclease activity as compared to the protein, wherein the CuPNF&#95;MCL showed a threefold greater nuclease activity as compared to the ZIF8&#95;MCL. The greater nuclease activity of CuPNF&#95;MCL is attributable to its mesoporous nature with higher pore size and pore volume as compared to that in case of ZIF8&#95;MCL, which is microporous in nature. Thus, in this paper, we have purified a nuclease like lectin from bitter gourd seeds and improved its nuclease property by converting it into inorganic hybrid nanomaterial of two types wherein higher activity was observed in the material having better porosity and surface area characteristics., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5'-TG-3' PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)