T-DNA Research Articles

Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging

The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.

Development of Tris‐(triazolyl)‐s–triazine Linker‐Based Cationic Amphiphiles: Stability and Gene Delivery Applications

AbstractThe ability to readily be designed, manufactured, and described has made cationic lipids one of the highly adaptable vehicles for the transfer of DNA, RNA, and numerous additional therapeutic compounds. In this article, we synthesized a variety of Tris‐(triazolyl)‐s‐triazine linker‐based cationic lipids with different types of anchoring groups, and assessed the transfection efficacy. Specifically, two identical tails like either two alpha‐tocopherol domains or cholesterol domains or aliphatic chains (saturated), and on the other hand, two different tails, alpha‐tocopherol domain and cholesterol domain or alpha‐tocopherol domain and aliphatic chain (saturated) were employed as hydrophobic motifs to synthesize the cationic lipids. The stability, size, and surface charge of liposomes were performed by dynamic light scattering technique. Cytotoxicity study on cell survival showed that the synthesized lipids are less hazardous to the cells despite the fact that cationic lipids have large molecular structure. The lipid having di alpha‐tocopherol domains demonstrated phenomenal β‐galactosidase protein expression and enhanced green fluorescence expression in the hepatocellular carcinoma cell line. Findings from the physico‐chemical and biological evaluations the developed lipids are compatible in various forms while maintaining low cytotoxicity and efficient carriers for nucleic acid delivery.

Self- and non-self-DNA on hands and sleeve cuffs.

Studying DNA transfer and persistence has become increasingly important over the last decade, due to the impressive sensitivity of modern DNA detection methods in forensic genetics. To improve our understanding of background DNA that could also potentially be transferred, we analyzed the DNA composition on the outside of sleeve cuffs and sampled DNA directly from the hands of four different collaborators upon their arrival at work during 25 working days. Sampling of their hands was repeated after several hours working in our department. The shedder status of the participants, as assumed from previous internal studies, was well re-produced in the study. However, we noticed that the DNA shedding capacity could also change drastically during the day, with one participant showing a more than sixfold increase between hands sampled in the morning and hands sampled in the afternoon. As expected, poor DNA shedders carry more relative amounts of non-self-DNA on their hands than good shedders. Non-self-alleles were detected in 95% of the samples. We also observed potential effects of hand washing and the mode of transport to get to work on the DNA amount. People living with family members occasionally carried their DNA on their hands and more frequently on their sleeve cuffs. Sleeve cuffs, as being close to our hands, have a large potential to transfer DNA from one place to another, yet they have sparsely been studied as DNA transfer intermediates so far. In general, we collected consistently more DNA from the sleeve cuffs than from the hands of the participants, demonstrating their importance as potential transfer vectors. More DNA was recovered from sleeve cuffs made of synthetic fabric than from cuffs made of cotton or leather. In the afternoon, DNA from co-habitant family members could not be detected on the hands anymore and the detection of profiles from colleagues became more frequent. From two out of 100 analyzed sleeve cuffs and two out of 200 sampled hands, we established unknown major DNA profiles that would have been suitable for an entry in the national DNA database. This finding demonstrates the possibility to transfer DNA that has most likely been picked up somewhere in the public space.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Intrageneric structural variation in organelle genomes from the genus Dystaenia (Apiaceae): genome rearrangement and mitochondrion-to-plastid DNA transfer.

During plant evolution, intracellular DNA transfer (IDT) occurs not only from organelles to the nucleus but also between organelles. To further comprehend these events, both organelle genomes and transcriptomes are needed. In this study, we constructed organelle genomes and transcriptomes for two Dystaenia species and described their dynamic IDTs between their nuclear and mitochondrial genomes, or plastid and mitochondrial genomes (plastome and mitogenome). We identified the putative functional transfers of the mitochondrial genes 5' rpl2, rps10, rps14, rps19, and sdh3 to the nucleus in both Dystaenia species and detected two transcripts for the rpl2 and sdh3 genes. Additional transcriptomes from the Apicaceae species also provided evidence for the transfers and duplications of these mitochondrial genes, showing lineage-specific patterns. Intrageneric variations of the IDT were found between the Dystaenia organelle genomes. Recurrent plastid-to-mitochondrion DNA transfer events were only identified in the D. takeshimana mitogenome, and a pair of mitochondrial DNAs of plastid origin (MIPTs) may generate minor alternative isoforms. We only found a mitochondrion-to-plastid DNA transfer event in the D. ibukiensis plastome. This event may be linked to inverted repeat boundary shifts in its plastome. We inferred that the insertion region involved an MIPT that had already acquired a plastid sequence in its mitogenome via IDT. We propose that the MIPT acts as a homologous region pairing between the donor and recipient sequences. Our results provide insight into the evolution of organelle genomes across the family Apiaceae.

Non-covalent binding interaction of bioactive coumarin esculetin with calf thymus DNA and yeast transfer RNA: A detailed investigation to decipher the binding affinities, binding location, interacting forces and structural alterations at a molecular level

Esculetin is a well-known coumarin derivative found abundantly in nature possessing an extensive array of pharmacological and therapeutic properties. Consequently, to comprehend its molecular recognition mechanism, our objective is to conduct a complete investigation of its interactions with the nucleic acid, specifically ct-DNA, and t-RNA, using spectroscopic and computational techniques. The intrinsic fluorescence of esculetin is quenched when it interacts with ct-DNA and t-RNA, and this occurs through a static quenching mechanism. The thermodynamic parameters demonstrated that the interaction is influenced by hydrogen bonding and weak van der Waals forces. CD and FT-IR results revealed no conformational changes in ct-DNA and t-RNA structure on binding with esculetin. Furthermore, competitive displacement assay with ethidium bromide, melting temperature, viscosity measurement, and potassium iodide quenching experiments, reflected that esculetin probably binds to the minor groove of ct-DNA. The molecular docking results provided further confirmation for the spectroscopic findings, including the binding location of esculetin and binding energies of esculetin complexes with ct-DNA and t-RNA. Molecular dynamics simulation studies demonstrated the conformational stability and flexibility of nucleic acids.

Electric organ discharge from electric eel facilitates DNA transformation into teleost larvae in laboratory conditions

Background Electric eels (Electrophorus sp.) are known for their ability to produce electric organ discharge (EOD) reaching voltages of up to 860 V. Given that gene transfer via intense electrical pulses is a well-established technique in genetic engineering, we hypothesized that electric eels could potentially function as a gene transfer mechanism in their aquatic environment. Methods To investigate this hypothesis, we immersed zebrafish larvae in water containing DNA encoding the green fluorescent protein (GFP) and exposed them to electric eel’s EOD. Results and Discussion Some embryos exhibited a mosaic expression of green fluorescence, in contrast to the control group without electrical stimulation, which showed little distinct fluorescence. This suggests that electric eel EOD has the potential to function as an electroporator for the transfer of DNA into eukaryotic cells. While electric eel EOD is primarily associated with behaviors related to sensing, predation, and defense, it may incidentally serve as a possible mechanism for gene transfer in natural environment. This investigation represents the initial exploration of the uncharted impact of electric eel EOD, but it does not directly establish its significance within the natural environment. Further research is required to understand the ecological implications of this phenomenon.

Opportunistic Bacteria of Grapevine Crown Galls Are Equipped with the Genomic Repertoire for Opine Utilization.

Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall-specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.

Indirect DNA Transfer and Forensic Implications: A Literature Review.

Progress in DNA profiling techniques has made it possible to detect even the minimum amount of DNA at a crime scene (i.e., a complete DNA profile can be produced using as little as 100 pg of DNA, equivalent to only 15-20 human cells), leading to new defense strategies. While the evidence of a DNA trace is seldom challenged in court by a defendant's legal team, concerns are often raised about how the DNA was transferred to the location of the crime. This review aims to provide an up-to-date overview of the experimental work carried out focusing on indirect DNA transfer, analyzing each selected paper, the experimental method, the sampling technique, the extraction protocol, and the main results. Scopus and Web of Science databases were used as the search engines, including 49 papers. Based on the results of this review, one of the factors that influence secondary transfer is the amount of DNA shed by different individuals. Another factor is the type and duration of contact between individuals or objects (generally, more intimate or prolonged contact results in more DNA transfer). A third factor is the nature and quality of the DNA source. However, there are exceptions and variations depending on individual characteristics and environmental conditions. Considering that secondary transfer depends on multiple factors that interact with each other in unpredictable ways, it should be considered a complex and dynamic phenomenon that can affect forensic investigation in various ways, for example, placing a subject at a crime scene who has never been there. Correct methods and protocols are required to detect and prevent secondary transfer from compromising forensic evidence, as well as the correct interpretation through Bayesian networks. In this context, the definition of well-designed experimental studies combined with the use of new forensic techniques could improve our knowledge in this challenging field, reinforcing the value of DNA evidence in criminal trials.

Overall Proportion of Total DNA Consistent with an Individual Briefly Handling a Firearm.

In forensic investigations, DNA profiles are routinely obtained from firearms evidence and alternative hypotheses may be proposed for consideration on the activity level. DNA profiles found to be consistent with the DNA profile of a specific individual could be a result of directly handling the firearm or other modes of transfer of DNA. Sixteen law-enforcement-owned firearms were evaluated with samples collected from the frame and slide area, the trigger and trigger guard area, and the front and rear sights after brief handling by laboratory personnel. Twenty-two out of forty-eight samples resulted in DNA profiles suitable for comparison, of which six resulted in likelihood ratios (LR) that demonstrated support for the hypothesis that included the brief handler as a contributor to the DNA profile obtained from the sample. Five of these samples were obtained from the frame and slide and one was from the trigger and trigger guard area. None of the DNA profiles obtained from the sights supported the inclusion of the brief handler as a contributor to the DNA profile. Gaining knowledge and supporting data on the nature of DNA profiles typically obtained from both owners and brief handlers can be useful for the purposes of evaluative reporting when considering results obtained from firearm evidence.

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria.

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established that the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that while most transfer events occur between cells in direct contact, the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a unique model for conjugation that revises our understanding of the DNA transfer mechanism and the dissemination of drug resistance and virulence genes within complex bacterial communities.

Chromosome-Level Genome Assembly for the Angiosperm Silene conica.

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on Pacific Bioscience HiFi, Hi-C, and Bionano technologies. The assembly produced 10 scaffolds (1 per chromosome) with a total length of 862 Mb and only ∼1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genus's ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology.

Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN-based toolbox was systematically expanded, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, two EcN-based technologies were developed, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, the EcN-based applications were further expanded by developing EcN native integrase-mediated genetic engineering and establishing an in vitro cell-free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating and making full use of EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Role of natural transformation in the evolution of small cryptic plasmids in Synechocystis sp. PCC 6803.

Small cryptic plasmids have no clear effect on the host fitness and their functional repertoire remains obscure. The naturally competent cyanobacterium Synechocystis sp. PCC 6803 harbours several small cryptic plasmids; whether their evolution with this species is supported by horizontal transfer remains understudied. Here, we show that the small cryptic plasmid DNA is transferred in the population exclusively by natural transformation, where the transfer frequency of plasmid-encoded genes is similar to that of chromosome-encoded genes. Establishing a system to follow gene transfer, we compared the transfer frequency of genes encoded in cryptic plasmids pCA2.4 (2378 bp) and pCB2.4 (2345 bp) within and between populations of two Synechocystis sp. PCC 6803 labtypes (termed Kiel and Sevilla). Our results reveal that plasmid gene transfer frequency depends on the recipient labtype. Furthermore, gene transfer via whole plasmid uptake in the Sevilla labtype ranged among the lowest detected transfer rates in our experiments. Our study indicates that horizontal DNA transfer via natural transformation is frequent in the evolution of small cryptic plasmids that reside in naturally competent organisms. Furthermore, we suggest that the contribution of natural transformation to cryptic plasmid persistence in Synechocystis is limited.

Deletions within intronic T-DNA lead to reversion of T-DNA mutant phenotypes.

Agrobacterium-mediated transformation enables random transfer-DNA (T-DNA) insertion into plant genomes. T-DNA insertion into a gene's exons, introns or untranscribed regions close to the start or stop codon can disrupt gene function. Such T-DNA mutants have been useful for reverse genetics analysis, especially in Arabidopsis thaliana. As T-DNAs are inserted into genomic DNA, they are generally believed to be stably inherited. Here, we report a phenomenon of reversion of intronic T-DNA mutant phenotypes. From a suppressor screen using intronic T-DNA pi4kβ1,2 double mutant, we recovered intragenic mutants of pi4kβ1, which suppressed the autoimmunity of the double mutant. These mutants carried deletions in the intronic T-DNAs, resulting in elevated transcription of normal PI4Kβ1. Such reversion of T-DNA insertional mutant phenotype stresses the need for caution when using intronic T-DNA mutants and reiterates the importance of using irreversible null mutant alleles in genetic analyses.

The Language of Life: A Game-Based Workshop for Introducing Protein Biosynthesis

Protein biosynthesis is a complex process that involves the transcription of DNA into mRNA and the subsequent translation of mRNA into proteins according to the genetic code. To introduce this fundamental process to a broad audience, we developed "The Language of Life", a game-based workshop that was presented at the Genoa Science Festival 2022, the largest science communication event in Italy. The game-based workshop employed jigsaw puzzle-like elements to represent DNA, mRNA, and aminoacyl transfer RNAs, enabling participants to pair them through codon combinations. The game-based workshop lasted for an hour and the framing was a "special mission" inside a cell. It consisted of an initial training phase that incorporated videos, models, and explanations, followed by practical "missions'' in which participants reproduce transcription and translation mechanisms by moving inside the cell and using the provided materials. The workshop was attended by 1,505 participants, primarily students aged 6-18, and received positive feedback. In this paper, we present our experience conducting this workshop and discuss its impact and potential for future use.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

Determination of the Impact of High-Intensity Pulsed Electromagnetic Fields on the Release of Damage-Associated Molecular Pattern Molecules.

High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by an externally applied pulsed magnetic field. Recently, HI-PEMF has been successfully used in the transfer of plasmid DNA and siRNA in vivo, with no or minimal infiltration of immune cells. In addition to gene electrotransfer, treatment with HI-PEMF has also shown potential for electrochemotherapy, where activation of the immune response contributes to the treatment outcome. The immune response can be triggered by immunogenic cell death that is characterized by the release of damage-associated molecular patterns (DAMPs) from damaged or/and dying cells. In this study, the release of the best-known DAMP molecules, i.e., adenosine triphosphate (ATP), calreticulin and high mobility group box 1 protein (HMBG1), after HI-PEMF treatment was investigated in vitro on three different cell lines of different tissue origin and compared with conventional electroporation treatment parameters. We have shown that HI-PEMF by itself does not cause the release of HMGB1 or calreticulin, whereas the release of ATP was detected immediately after HI-PEMF treatment. Our results indicate that HI-PEMF treatment causes no to minimal release of DAMP molecules, which results in minimal/limited activation of the immune response.