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Self‐inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species‐specific growth inhibitor in plants and proposed as an explanation of negative plant–soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self‐inhibition processes. Moreover, the existence of a general species‐specific biological effect of exDNA raises interesting questions on its possible involvement in self‐recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.

The capacity to actively release genetic material into the extracellular environment has been reported for bacteria, archaea, fungi, and in general, for microbial communities, but it is also described in the context of multicellular organisms, animals and plants. This material is often present in matrices that locate outside the cells. Extracellular matrices have important roles in defense response and disease in microbes, animal and plants cells, appearing as barrier against pathogen invasion or for their recognition. Specifically, neutrophils extracellular traps (NETs) in animals and root extracellular traps (RETs) in plants, are recognized to be important players in immunity. A growing amount of evidence revealed that the extracellular DNA, in these contexts, plays an active role in the defense action. Moreover, the protective role of extracellular DNA against antimicrobials and mechanical stress also appears to be confirmed in bacterial biofilms. In parallel, recent efforts highlighted different roles of self (homologous) and non-self (heterologous) extracellular DNA, paving the way to discussions on its role as a “Damage-associated molecular pattern” (DAMP). We here provide an evolutionary overview on extracellular DNA in extracellular matrices like RETs, NETs, and microbial biofilms, discussing on its roles and inferring on possible novel functionalities.

Stressed organisms identify intracellular molecules released from damaged cells due to trauma or pathogen infection as components of the innate immune response. These molecules called DAMPs (Damage-Associated Molecular Patterns) are extracellular ATP, sugars, and extracellular DNA, among others. Animals and plants can recognize their own DNA applied externally (self-exDNA) as a DAMP with a high degree of specificity. However, little is known about the microalgae responses to damage when exposed to DAMPs and specifically to self-exDNAs. Here we compared the response of the oilseed microalgae Neochloris oleoabundans to self-exDNA, with the stress responses elicited by nonself-exDNA, methyl jasmonate (MeJA) and sodium bicarbonate (NaHCO3). We analyzed the peroxidase enzyme activity related to the production of reactive oxygen species (ROS), as well as the production of polyphenols, lipids, triacylglycerols, and phytohormones. After 5 min of addition, self-exDNA induced peroxidase enzyme activity higher than the other elicitors. Polyphenols and lipids were increased by self-exDNA at 48 and 24 h, respectively. Triacylglycerols were increased with all elicitors from addition and up to 48 h, except with nonself-exDNA. Regarding phytohormones, self-exDNA and MeJA increased gibberellic acid, isopentenyladenine, and benzylaminopurine at 24 h. Results show that Neochloris oleoabundans have self-exDNA specific responses.

cGAS-STING pathway stands at the forefront of innate immunity and plays a critical role in regulating adaptive immune responses, making it as a key orchestrator of anti-tumor immunity. Despite the great potential, clinical outcomes with cGAS-STING activators have been disappointing due to their unfavorable in vivo fate, signaling an urgent need for innovative solutions to bridge the gap in clinical translation. Recent advancements in nanotechnology have propelled cGAS-STING-targeting nanomedicines to the cutting-edge of cancer therapy, leveraging precise drug delivery systems and multifunctional platforms to achieve remarkable region-specific biodistribution and potent therapeutic efficacy. In this review, we provide an in-depth exploration of the molecular mechanisms that govern cGAS-STING signaling and its potential to dynamically modulate the anti-tumor immune cycle. We subsequently introduced several investigational cGAS-STING-dependent anti-tumor agents and summarized their clinical trial progress. Additionally, we provided a comprehensive review of the unique advantages of cGAS-STING-targeted nanomedicines, highlighting the transformative potential of nanotechnology in this field. Furthermore, we comprehensively reviewed and comparatively analyzed the latest breakthroughs cGAS-STING-targeting nanomedicine, focusing on strategies that induce cytosolic DNA generation via exogenous DNA delivery, chemotherapy, radiotherapy, or dynamic therapies, as well as the nanodelivery of STING agonists. Lastly, we discuss the future prospects and challenges in cGAS-STING-targeting nanomedicine development, offering new insights to bridge the gap between mechanistic research and drug development, thereby opening new pathways in cancer treatment.

BackgroundCurrent antibody-drug conjugates (ADCs) face limitations due to a lack of tumor-selective targets, inefficient internalization, and challenges in reaching tumors in challenging sites, ultimately limiting their therapeutic efficacy. We developed and characterized V66-exatecan, a novel ADC composed of V66, a humanized antibody with high affinity for extracellular DNA (exDNA), conjugated to exatecan via a cleavable linker. This ADC employs a dual-targeting mechanism based on exDNA and ENT2 transporter expression to enhance nuclear drug delivery and tumor specificity. This study evaluates its anti-tumor activity, mechanism of action, ability to treat challenging tumors, and safety profile.MethodsTo validate tumor selectivity, V66 or a control antibody were conjugated to a fluorescent tag and injected intravenously into tumor-bearing mice; biodistribution analysis demonstrated selective accumulation in tumors and nuclear localization within tumor cells. V66 was then conjugated to exatecan via a cleavable linker. In vitro assays across diverse cancer cell lines assessed cytotoxicity, DNA damage response (DDR) activation, and TOP1 degradation. In vivo efficacy was evaluated in xenograft models of triple-negative breast cancer (TNBC) and BRCA1/2-deficient tumors, including intracranial medulloblastoma. These models were used to assess tumor growth inhibition, survival benefit, and blood-brain barrier (BBB) permeability. Toxicity was assessed through a dose-escalation study, with analysis of hematologic parameters, histopathology of major organs, and liver and kidney function tests (ALT, AST, BUN, total protein) following short- and long-term treatment.ResultsV66-exatecan demonstrated potent anti-tumor activity in multiple cancer cell lines but not on healthy mouse primary fibroblasts, with EC50 values in the low nanomolar range. It induced robust DDR signaling, TOP1 degradation, and bystander killing effects. BRCA1/2-deficient models exhibited enhanced penetration and sensitivity, with up to 17-fold lower EC50 compared to BRCA-proficient controls. In vivo, V66-exatecan significantly inhibited tumor growth and extended survival in both TNBC and BRCA-mutant CNS tumors, including complete regressions and prolonged median survival in BRCA2-deficient models. Toxicology studies revealed no significant hematologic, renal, hepatic, or bone marrow toxicity, even at high or repeated doses.ConclusionsV66-exatecan represents a next-generation of ADCs that overcomes key limitations of traditional platforms by exploiting exDNA-driven tumor selectivity and ENT2-mediated nuclear delivery. It demonstrates broad therapeutic efficacy and a favorable safety profile, supporting its potential for treating DDR-deficient and hard-to-reach tumors.

The inhibitory effect of extracellular DNA (exDNA) on the growth of conspecific individuals was demonstrated in different kingdoms. In plants, the inhibition has been observed on root growth and seed germination, demonstrating its role in plant–soil negative feedback. Several hypotheses have been proposed to explain the early response to exDNA and the inhibitory effect of conspecific exDNA. We here contribute with a whole-plant transcriptome profiling in the model species Arabidopsis thaliana exposed to extracellular self- (conspecific) and nonself- (heterologous) DNA. The results highlight that cells distinguish self- from nonself-DNA. Moreover, confocal microscopy analyses reveal that nonself-DNA enters root tissues and cells, while self-DNA remains outside. Specifically, exposure to self-DNA limits cell permeability, affecting chloroplast functioning and reactive oxygen species (ROS) production, eventually causing cell cycle arrest, consistently with macroscopic observations of root apex necrosis, increased root hair density and leaf chlorosis. In contrast, nonself-DNA enters the cells triggering the activation of a hypersensitive response and evolving into systemic acquired resistance. Complex and different cascades of events emerge from exposure to extracellular self- or nonself-DNA and are discussed in the context of Damage- and Pathogen-Associated Molecular Patterns (DAMP and PAMP, respectively) responses.

PREMISE OF THE STUDY: Root border cells are programmed to separate from the root cap as it penetrates the soil environment, where the cells actively secrete >100 extracellular proteins into the surrounding mucilage. The detached cells function in defense of the root tip by an extracellular trapping process that also requires DNA, as in mammalian white blood cells. Trapping in animals and plants is reversed by treatment with DNase, which results in increased infection. The goal of this study was to evaluate the role of DNA in the structural integrity of extracellular structures released as border cells disperse from the root tip upon contact with water. METHODS: DNA stains including crystal violet, toluidine blue, Hoechst 33342, DAPI, and SYTOX green were added to root tips to visualize the extracellular mucilage as it absorbed water and border cell populations dispersed. DNase I was used to assess structural changes occurring when extracellular DNA was degraded. KEY RESULTS: Complex masses associated with living border cells were immediately evident in response to each stain, including those that are specific for DNA. Treating with DNase I dramatically altered the appearance of the extracellular structures and their association with border cells. No extracellular DNA was found in association with border cells killed by freezing or high‐speed centrifugation. This observation is consistent with the hypothesis that, as with border cell extracellular proteins, DNA is secreted by living cells. CONCLUSION: DNA is an integral component of border cell extracellular traps.

eDNA, also known as environmental DNA, has garnered significant attention due to its potential applications in various fields such as biodiversity assessment, species distribution monitoring, ecological interaction analysis, and quantitative analysis. However, the presence of non-selective DNA signals in eDNA samples poses challenges in accurately detecting species, assessing biodiversity, and conducting quantitative analysis. To address these limitations, this study developed a novel method for selectively detecting iDNA from specific species in eDNA samples. The method involved the application of PMA treatment to Alexandrium spp. effectively preventing the detection of non-selective exDNA signals. Additionally, by optimizing the filter size used in the sampling process, the researchers were able to selectively collect and analyze iDNA from species of interest, particularly Alexandrium spp. Furthermore, the study successfully demonstrated the selective collection and analysis of iDNA from Alexandrium spp. cysts present in the sediment layer, further strengthening the findings. The results indicated that the combined use of PMA treatment and filter size optimization significantly enhanced the selective detection capability of iDNA. The successful selective detection of iDNA from eDNA in the sediment layer highlights the practical applicability of the developed method. This study holds promise for advancing eDNA monitoring technology by providing a selective iDNA detection method utilizing PMA. Moreover, these findings lay the foundation for effectively utilizing iDNA in environmental conservation, monitoring, and ecological research.

Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of Glycine max using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete Phytophthora parasitica early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.

Background As roots penetrate soil, specialized cells called 'border cells' separate from root caps and contribute a large proportion of exudates forming the rhizosphere. Their function has been unclear. Recent findings suggest that border cells act in a manner similar to that of white blood cells functioning in defense. Histone-linked extracellular DNA (exDNA) and proteins operate as 'neutrophil extracellular traps' to attract and immobilize animal pathogens. DNase treatment reverses trapping and impairs defense, and mutation of pathogen DNase results in loss of virulence. Scope Histones are among a group of proteins secreted from living border cells. This observation led to the discovery that exDNA also functions in defense of root caps. Experiments revealed that exDNA is synthesized and exported into the surrounding mucilage which attracts, traps and immobilizes pathogens in a host-microbe specific manner. When this plant exDNA is degraded, the normal resistance of the root cap to infection is abolished. Conclusions Research to define how exDNA may operate in plant immunity is needed. In the meantime, the specificity and stability of exDNA and its association with distinct microbial species may provide an important new tool to monitor when, where, and how soil microbial populations become established as rhizosphere communities.