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Background: Exosomes are cell-derived vesicles and bear a specific set of nucleic acids including DNA (exoDNA). Thus, this study is to explore whether exoDNA in malignant pleural effusions (MPEs) could be a novel DNA source for mutation detection of epidermal growth factor receptor (EGFR).Methods: In this study, 52 lung adenocarcinoma patients were enrolled, and EGFR mutation status was detected with tumor tissues as well as cell blocks and exosomes in MPEs. The sensitivity, specificity and consistency of EGFR detection using exosomes were evaluated, compared with gene detection using tumor tissues and cell blocks. And the clinical response of patients who were detected as EGFR mutation in exosomes and treated with EGFR tyrosine kinase inhibitor (EGFR-TKI) was explored.Results: Gene detection using exosomes showed sensitivity of 100%, specificity of 96.55% and coincidence rate of 98.08% (Kappa = 0.961, P < 0.001), compared with detection using tumor tissues and cell blocks. After EGFR-TKI treatment, patients detected as EGFR mutation by exosomes showed efficacy rate of 83% and disease control rate of 100%. And patients who were detected as wild type in tumor tissues or cell blocks but EGFR mutation in exosomes turned up as PR or SD.Conclusions: These results demonstrated that exoDNA in MPEs could be used as a DNA source for EGFR detection in lung adenocarcinoma.

Single-molecule detection technology is a technique capable of detecting molecules at the single-molecule level, characterized by high sensitivity, high resolution, and high specificity. Nanopore technology, as one of the single-molecule detection tools, is widely used to study the structure and function of biomolecules. In this study, we constructed a small-sized nanopore with a pore-cavity-pore structure, which can achieve a higher reverse capture rate. Through simulation, we investigated the electrical potential distribution of the nanopore with a pore-cavity-pore structure and analyzed the influence of pore size on the potential distribution. Accordingly, different pore sizes can be designed based on the radius of gyration of the target biomolecules, restricting their escape paths inside the chamber. In the future, nanopores with a pore-cavity-pore structure based on two-dimensional thin film materials are expected to be applied in single-molecule detection research, which provides new insights for various detection needs., (© 2024 IOP Publishing Ltd.)

The aim of this study was to evaluate motility, viability, DNA integrity and apoptosis of spermatozoa when washed semen samples were kept for up to 12 days at 4–6°C and 25°C. In this experimental study, 26 normozoospermic semen samples were washed twice in Modified Ham's F10 and resuspended in IVF fertilisation medium. Half of the specimens were stored at 4–6°C, and the other half was kept at 25°C for 12 days. The proportions of viable, motile, spermatozoa with double‐stranded DNA and apoptotic spermatozoa were examined during storage time. Apoptosis was measured using annexin V‐PI staining followed by flow cytometry. Results showed that sperm motility and viability decreased during 12 days of sample storage (p <.001). There was no significant difference between the two temperatures in terms of motility and viability for up to 2 days (p <.05). The percentage of spermatozoa with double‐stranded DNA remained unchanged during the 12 days of storage at both temperatures (p >.05). Although there was no difference between the two temperatures in terms of motility, viability and apoptosis during the first two days of storage, storage of spermatozoa at 4–6°C is better than storage for a longer period than storage at 25°C. Sperm DNA resisted against denaturation during storage. [ABSTRACT FROM AUTHOR]

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The extensional rheology of polymer melts and dilute polymer solutions has been extensively examined through experiments and theoretical predictions. However, a systematic study of the extensional rheology of polymer solutions in the semidilute regime, in terms of examining the effects of concentration and molecular weight, has not been carried out so far. Previous experimental studies of the shear rheology of semidilute polymer solutions have demonstrated that their behaviour is distinctively different from that observed in the dilute and concentrated regimes. This difference in behaviour is anticipated to be even more pronounced in extensional flows, which play a critical role in a number of industrial contexts such as fiber spinning and ink-jet printing. In this work, the extensional rheology of linear, double-stranded DNA molecules, spanning an order of magnitude of molecular weights (25-289 kilobasepairs) and concentrations (0.03-0.3 mg/ml), has been investigated. DNA solutions are now used routinely as model polymeric systems due to their near-perfect monodispersity. Measurements have been carried out with a filament stretching rheometer since it is the most reliable method for obtaining an estimate of the elongational stress growth of a polymer solution. Transient and steady-state uniaxial extensional viscosities of DNA dissolved in a solvent under excess salt conditions, with a high concentration of sucrose in order to achieve a sufficiently high solvent viscosity, have been determined in the semidilute regime at room temperature. The dependence of the steady state uniaxial extensional viscosity on molecular weight, concentration and extension rate is measured with a view to determining if data collapse can be observed with an appropriate choice of variables. Steady state shear viscosity measurements suggest that sucrose-DNA interactions might play a role in determining the observed rheological behaviour of semidilute DNA solutions with sucrose as a component in the solvent. [ABSTRACT FROM AUTHOR]

Background: Exosomes are cell-derived vesicles and bear a specific set of nucleic acids including DNA (exoDNA). Thus, this study is to explore whether exoDNA in malignant pleural effusions (MPEs) could be a novel DNA source for mutation detection of epidermal growth factor receptor (EGFR). Methods: In this study, 52 lung adenocarcinoma patients were enrolled, and EGFR mutation status was detected with tumor tissues as well as cell blocks and exosomes in MPEs. The sensitivity, specificity and consistency of EGFR detection using exosomes were evaluated, compared with gene detection using tumor tissues and cell blocks. And the clinical response of patients who were detected as EGFR mutation in exosomes and treated with EGFR tyrosine kinase inhibitor (EGFR-TKI) was explored. Results: Gene detection using exosomes showed sensitivity of 100%, specificity of 96.55% and coincidence rate of 98.08% (Kappa = 0.961, P < 0.001), compared with detection using tumor tissues and cell blocks. After EGFR-TKI treatment, patients detected as EGFR mutation by exosomes showed efficacy rate of 83% and disease control rate of 100%. And patients who were detected as wild type in tumor tissues or cell blocks but EGFR mutation in exosomes turned up as PR or SD. Conclusions: These results demonstrated that exoDNA in MPEs could be used as a DNA source for EGFR detection in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

The increasing use of pesticides is a major health issue. There is therefore a need for improved analytical methods and more knowledge on the interaction of pesticides with living organisms. Here, we developed the analysis of profluralin in water and soils by cyclic voltammetry, square-wave voltammetry and square-wave adsorptive stripping voltammetry. We also studied the interaction between profluralin and double-stranded DNA. Results of cyclic voltammetric studies indicate that the reduction processes have an irreversible and adsorption-like character, as a possible consequence of the reduction of nitro groups of profluralin. Quantification was found linear from 60 to 800 nmol L−1 with a detection limit of 17 nmol L−1 using square-wave adsorptive stripping voltammetry. Results of interactions with double-stranded DNA from salmon sperm revealed that profluralin forms a complex with DNA by electrostatic binding. Values of Gibbs free energy confirmed that formation of profluralin–DNA complex is spontaneous and favourable. [ABSTRACT FROM AUTHOR]

Highlights • A convenient ECL biosensing method for selective detection of 5-hmC was developed. • The method is based on thiolated anchor probe DNA to immobilize 5-hmC-dsDNA. • The method can be used for detection of 5-hmC in genomic DNA. Abstract Herein, a novel and convenient electrogenerated chemiluminescence (ECL) biosensing method for the quantitative detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA was developed by employing thiolated anchor probe DNA to immobilize 5-hydroxymethylcytosine double-stranded DNA (5-hmC-dsDNA) and a ruthenium(II) complex-tagged reporter probe as an ECL probe. Briefly, the 5-hmC-dsDNA was firstly oxidized to 5-formylcytosine dsDNA (5-fC-dsDNA) by KRuO 4 in high yields. Subsequently, the thiolated anchor probe DNA is employed to help immobilize the 5-fC-dsDNA onto the gold electrode surface via the Au-S bond. An ECL probe DNA, labeled with a signal transducer − Ru(bpy) 2 (cbpy)NHS, hybridized with the anchor probe DNA on the electrode, resulting in an intensive ECL emission. The obtained ECL intensity was linearly proportional to the content of 5-hmC in the range from 0.0090% to 0.5761%, with the limit of detection calculated to be 0.0036% (S/N = 3). Finally, we applied the developed 5-hmC detection strategy in various mice tissues. It demonstrates that the content of 5-hmC measured by this method in different mice tissues vary significantly: 5-hmC is highest in brain and lowest in spleen. The success in assaying the mice tissues samples signifies its great promise in a broad range of practical applications for the quantitative detection of 5-hmC in genomic DNA. [ABSTRACT FROM AUTHOR]

Previously, we reported on the development of a therapeutic regimen allowing eradication of primary murine Krebs-2 ascites transplants. This protocol involved multiple injections of dsDNA preparations administered during the NER and HR phases of repair of interstrand DNA cross-links induced by prior cyclophosphamide treatments. Mice treated under this protocol frequently developed secondary ascites, which indicated that some tumor-inducing cancer stem cells could survive the treatment and caused relapse. Further, we observed that animals receiving multiple dsDNA injections developed pronounced systemic inflammatory response. This prompted us to develop a more straightforward treatment regimen based on the synergistic activity of cyclophosphamide and dsDNA preparations, which would allow complete eradication of established primary Krebs-2 ascites and also be less toxic for the treated animals. This protocol relies on a precisely timed single injection of dsDNA during the NER/HR transition period of each repair cycle. Under this protocol, 8-day remission of Krebs-2 engrafted mice was achieved, which was similar to the results of the multiple-injection treatment schedule. We observed an increase in the average life span of Krebs-2- transplanted mice on a single-injection regimen, which was consistent with reduced toxicity of such treatment.

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2 ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.

Existence of a small subset of cancer cells referred to as tumor initiating stem cells (TISCs) largely responsible for tumor progression and resistance to chemotherapeutic cytostatic drugs reperesent an important recent paradigm shift. The present work is the first report in the series of papers from our group where we describe the development of anticancer therapy based on the selective targeting of TISCs. Here were characterize a cytoreductive activity of cyclophosphamide (CP), double-stranded DNA (dsDNA) and combinations thereof against the TISC population present in mouse Krebs-2 ascites. We evaluated engraftment potential of Krebs-2 cancer cells treated in ascites-bearing mice in vivo, followed by re-engraftment to congenic recipient mice in a form of a solid graft. These data indicate that with our approach TISCs can be completely eliminated even from a well-established ascites. We demonstrate that dsDNA-internalizing and CD34-positive cells are more sensitive to the synergistic effects of CP and dsDNA. When Krebs-2 ascites are treated with human DNA 1-12 hours post CP injection, this results in either elimination of cells that internalize TAMRA-labeled DNA (TISCs) or alters their phenotype, which is accompanied with the loss of surface expression of CD34. Next, we show that the timepoint 18 hrs post CP treatment is critical to the ongoing repair process in that it divides the repair into two phases: nucleotide excision repair + dsDNA break repair and homologous recombination. Importantly, both of these phases can be conveniently used for targeting the tumorigenic potential of the graft. In the context of monotherapy, CP is most effective against ascites grafts when administered as serial injections. To achieve maximum efficiency, the timing of consecutive injections must match the time when cancer cells found at G2/M during the first injection enter G1/S and/or the time of active repair via homologous recombination.

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites.

Electron-microscopic analysis of the ultrastructure of the Krebs-2 carcinoma ascites cells in the first 90 min immediately after their exposure to fragmented double-stranded DNA has been performed. Morphological attributes of the treated cancer cells indicate the induction in these cells of destructive processes of presumably apoptotic type. The predominance of dystrophic-destructive changes in cells after the addition of DNA is supposed to be a consequence of the disturbance in metabolic processes caused by the experimental action. [ABSTRACT FROM AUTHOR]

The experimental phase determination of crystal structures of nucleic acids and nucleic acid–ligand complexes would benefit from a facile method. Even for double‐stranded DNA, software‐generated models are generally insufficiently accurate to serve as molecular replacement search models, necessitating experimental phasing. Here, it is demonstrated that Zn2+ ions coordinated to the N7 atom of guanine bases generate sufficient anomalous signal for single‐wavelength anomalous diffraction (SAD) phasing of DNA crystal structures. Using zinc SAD, three crystal structures of double‐stranded DNA oligomers, 5′‐AGGGATCCCT‐3′, 5′‐GGGATCCC‐3′ and 5′‐GAGGCCTC‐3′, were determined. By determining the crystal structure of one of these oligomers, GAGGCCTC, in the presence of Mg2+ instead of Zn2+, it was demonstrated that Zn2+ is not structurally perturbing. These structures allowed the analysis of structural changes in the DNA on the binding of analogues of the natural product mithramycin to two of these oligomers, AGGGATCCCT and GAGGCCTC. Zinc SAD may become a routine approach for determining the crystal structures of nucleic acids and their complexes with small molecules. Guanine N7–Zn2+ coordination served as the basis for the determination of three DNA crystal structures using zinc single‐wavelength anomalous diffraction. This method is likely to be broadly applicable to the experimental determination of crystal structures of nucleic acids. [ABSTRACT FROM AUTHOR]

Kanamycin is an aminoglycoside antibiotic that can be useful against both gram negative and positive bacteria. However, if its serum levels are not controlled properly, it can cause serious side effects like ototoxicity and nephrotoxicity. The aim of this study was to design a simple and rapid fluorescent aptasensor for detection of kanamycin, based on Aptamer/Complementary strand (dsDNA)-capped mesoporous silica nanoparticles (MSNs) and Rhodamine B as a fluorescent probe. The MSNs pores were filled with Rhodamine B and then gated with dsDNA. In the presence of kanamycin, the aptamer sequence was separated from its complementary strand (CS), so that, uncovered the pores and leading to leakage of Rhodamine B. Thus, a significant increase in the fluorescence intensity was observed. The relative fluorescence intensity showed a linearity range from 24.75 nM to 137.15 nM of kanamycin with a detection limit of 7.5 nM. The aptasensor also showed to be useful for detection of kanamycin in serum samples and was able to distinguish kanamycin from other antibiotics, resulting in a sensitive, rapid and inexpensive method for kanamycin detection. [ABSTRACT FROM AUTHOR]

This study aims at assessing NF-kB activity in unstable angina (UA) patients free of symptoms after a 1 year follow-up (1YFU). Plasma oxidized low-density lipoproteins (oxLDL), circulating NF-kB, Interleukin 6 (IL-6) and Interleukin 1β (IL-1β), high-sensitivity C-reactive protein (hs-CRP), as markers of oxidative stress and inflammation and plasma double-stranded DNA (ds-DNA), as marker of Neutrophil Extracellular Traps (NETs), were measured in 23 of the previously enrolled 27 UA patients. These measurements were compared to the UA data at baseline, and then compared to the data derived from the stable angina (SA) and controls (C) enrolled in our previous study (we demonstrated that UA had higher levels of NF-kB compared to SA and C). After a 1YFU, UA patients show a significant decrease in NF-kB, IL-6, hs-CRP, oxLDL, and ds-DNA plasma levels (p < 0.001) and in IL-1β and White Blood Cells (WBC) (p < 0.005), without differences in lipid and glucose assessment. If compared to SA and C, UA after a 1YFU have higher levels of NF-kB, IL-6, ds-DNA, WBC, and oxLDL compared to C (p < 0.001), but only IL-6 is higher than SA (p < 0.001). No differences are found in lipid and glucose assessment. After a 1YFU, patients with a history of UA improve their oxidative and inflammatory status, such as the levels of circulating ds-DNA, without achieving the status of C. They become comparable to SA subjects. This study provides new insight on the multiple and apparently contradictory facets of NF-kB in UA and on its possible role as mediator in NETs’ formation. [ABSTRACT FROM AUTHOR]

Abstract: Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi–Goutières Syndrome (AGS). The recently discovered cyclic‐GMP‐AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1‐deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self‐DNA. Collectively, these findings might lead to the development of new therapeutics against IFN‐driven pathologies due to cGAS activation. [ABSTRACT FROM AUTHOR]

Recent progress in the combination of nucleic acids and carbon nanotubes (CNTs) has been briefly reviewed here. Since discovering the hybridization phenomenon of DNA molecules and CNTs in 2003, a large amount of fundamental and applied research has been carried out. Among thousands of papers published since 2003, approximately 240 papers focused on biological applications were selected and categorized based on the types of nucleic acids used, but not the types of CNTs. This survey revealed that the hybridization phenomenon is strongly affected by various factors, such as DNA sequences, and for this reason, fundamental studies on the hybridization phenomenon are important. Additionally, many research groups have proposed numerous practical applications, such as nanobiosensors. The goal of this review is to provide perspective on biological applications using hybrids of nucleic acids and CNTs.

The nonlinear rheological behaviors of concentrated double-stranded (ds) and single-stranded (ss) DNA solutions were studied under standard oscillatory and large amplitude oscillatory shear (LAOS) deformations. To decompose the total stress into the elastic and viscous components, the raw nonlinear waveforms were analyzed using the MITlaos software package. We found that smooth and rigorous intracycle strain stiffening and shear thinning dominate the elastic and viscous decomposition, respectively, of the ds DNA sample. In contrast, the ss DNA sample exhibits initial intracycle strain stiffening, followed by a transition region and a terminal flow behavior. Insights into the nonlinear response of the DNA solutions were achieved by performing particle image velocimetry (PIV) at a wide range of imposed strain amplitudes and two angular frequencies. The rheo-PIV results of the ds DNA sample show a substantially modified flow field with distinct strain-driven shear bands at both angular frequencies examined. The bands formed by the ss DNA sample were weak even at the maximum strain amplitude. The results from this work are insightful from a traditional bulk rheological perspective, as well as for further single-molecular studies. [ABSTRACT FROM AUTHOR]

The ability to directly detect double-stranded DNA (dsDNA) without sequence-preference continues to be a major challenge. Herein, we report an electrochemical method for the direct, highly sensitive detection of dsDNA based on the strand replacement of dsDNA by peptide nucleic acid (PNA) and the in situ growth of electroactive polymers through the surface-initiated electrochemically mediated atom transfer radical polymerization (SI-eATRP). Thiolated PNA molecules are firstly self-assembled onto gold electrode surface for the specific recognition of target dsDNA (dsDNA-T), which in turn leads to the formation of a high density of PNA/DNA heteroduplexes on the electrode surface for the subsequent attachment of ATRP initiators via the phosphate-Zr 4+ -carboxylate chemistry. By applying a negative potential to the electrode, the air-stable Cu II deactivators can be reduced into the Cu I activators so as to trigger the surface-initiated polymerization for the in situ growth of electroactive polymers. Due to the strand replacement of dsDNA by PNA, dsDNA can be directly detected without sequence-preference. Besides, the growth of polymers enables the modification of numerous electroactive probes, thereby greatly improving the electrochemical signal. Under optimal conditions, a good linearity between the electrochemical signal and the logarithm of dsDNA-T concentration over the range from 1.0 fM to 1.0 nM, with a detection limit of 0.47 fM, can be obtained. Results indicate that it is highly selective, and holds high anti-interference capability in the presence of human serum samples. Therefore, this method offers great promises in providing a universal and efficient solution for the direct detection of dsDNA. [ABSTRACT FROM AUTHOR]

• The ingress and egress kinetics of biomolecules studied in sub-3 μ m particles • A rigorous analytical solution is derived for a rectangular concentration change. • The kinetics are reported for mAb, dsDNA, and virus-like particles in fast UHPLC. • BEH Particles are out of equilibrium with mobile phase for dsDNA and viruses. • Egress kinetics is very slow suggesting using non-porous separation materials. The increasing demand for the characterization of large biomolecules such as monoclonal antibodies, double-stranded deoxyribonucleic acid (dsDNA), and virus-like particles (VLPs) is raising fundamental questions pertaining to their absorption (ingress) and escape (egress) kinetics from fully porous particles. The exact expression of their concentration profiles is derived as a function of time and radial position across a single sub-3 μ m Bridge-Ethylene-Hybrid (BEH TM) Particle present in size exclusion chromatography (SEC) columns. The boundary condition at the external surface area of the particle is a rectangular concentration profile mimicking the passage of the chromatographic zone. Four different BEH Particles were considered in the calculations depending on the molecular size of the analyte: 2.0 μ m 100 Å BEH Particles for small molecules, 2.0 μ m 200 Å BEH Particles for monoclonal antibodies, 2.0 μ m 300 Å BEH Particles for dsDNA (100 base pairs), and 2.5 μ m 900 Å BEH Particles for virus-like particles (VLPs). The calculated concentration profiles of small molecules and monoclonal antibodies confirm that all BEH Particles present in the column reach quasi-instantaneously thermodynamic equilibrium with the bulk mobile phase during the passage of the chromatographic band. This is no longer the case for larger biomolecules such as dsDNA or VLPs, especially when the SEC particle is located near the column inlet and for high velocities. The kinetics of biomolecule egress is slower than its kinetics of ingress leading to pronounced peak tailing. The mean concentration of the largest biomolecules in the SEC particles remains always smaller than the maximum bulk concentration. This persistent and transient intra-particle diffusion regime has direct implications on the theoretical expressions of the observed retention factors and plate heights. Classical theories of chromatography assume uniform spatial distribution of the analyte in the particle volume: this hypothesis is not verified for the largest biomolecules. These results imply that non-porous particles or monolithic structures are the most promising stationary phases for the separation and purification of the largest biomolecules in life science. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an extremely heterogenous autoimmune disorder. A key biomarker, the double stranded (ds) DNA autoantibody, provides diagnostic specificity for SLE. We analyzed anti-dsDNA by mass spectrometry (MS) to determine if ascertaining the autoantibody’s heavy chain variable region (IGHV) may hold any clinical relevance. A cross-sectional study of 32 SLE patients (75% female) in a single center was performed. Serum anti-dsDNA was subjected to MS analyses. Obtained IGHV subfamilies were correlated with active clinical features of SLE, as determined by medical record reviews. We established significant associations with the presence of IGHV3-15 and active neuropsychiatric lupus (relative risk [RR] 5.71); IGHV3-21, IGHV3-23 and IGHV4-34 and leukopenia (RR 13.70, 2.14 and 10.29 respectively); and IGHV3-23 and serositis (RR 2.41) and cutaneous lesions (RR 2.82). This study provides the first evidence for the clinical benefits of deep anti-dsDNA profiling through MS, and provides an avenue for improving predictive medicine for SLE patients. Future studies with a greater number of patients, and to determine if these subfamilies have direct pathogenic properties are required.

Coarse-grained models of DNA have made important contributions to the determination of the physical properties of genomic DNA, working as a molecular machine for gene regulation. In this study, to analyze the global dynamics of long DNA sequences with consideration of sequence-dependent geometry, we propose elastic network models of DNA where each particle represents k nucleotides (1-particle-per-k-nucleotides, 1PkN). The models were adjusted according to profiles of the anisotropic fluctuations obtained from our previous 1-particle-per-1-nucleotide (1P1N) model, which was proven to reproduce such profiles of all-atom models. We confirmed that the 1P3N and 1P4N models are suitable for the analysis of detailed dynamics such as local twisting motion. The models are intended for the analysis of large structures, e.g., 10-nm fibers in the nucleus, and nucleoids of mitochondrial or phage DNA at low computational costs. As an example, we surveyed the physical characteristics of the whole mitochondrial human and Plasmodium falciparum genomes. [ABSTRACT FROM AUTHOR]

Morphological changes in the thymus of female Wistar rats with experimental mammary gland carcinomas were studied. After adjuvant therapy, the area of the cortical matter and density of parenchymal cells in the thymus decreased, while areas of the medulla, connective tissue, and content of immunoblasts and macrophages increased. In the thymuses of rats receiving exogenous DNA, morphological signs of activation of the lymphoid and epithelial components were found: areas of the cortex and medulla, glandular and connective tissue corresponded to the values in intact animals, the counts of lymphocytes in the central part of the cortical matter and of macrophages in all zones of the thymus increased, and lymphocyte migration from the thymus increased (in comparison with the chemotherapy group). [ABSTRACT FROM AUTHOR]

Patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. So the assay of DNase I is of high importance. A colorimetric assay is described here for the determination of the activity of DNase. It is based on strand scission of dsDNA as catalyzed by DNase I. The products of digestion (nucleoside monophosphates) can better stabilize citrate capped AuNPs than dsDNA. In the absence of DNase I, the AuNPs aggregate in presence of NaCl and then display a blue color. In the presence of the analyte (DNase I), AuNPs do not aggregate but rather remain dispersed and display a red color. These findings were exploited to design a DNase I activity assay that is based on the measurement of ratio of absorbances at 520 nm (red) to 650 nm (blue). The detection limit for DNase I activity is found to be 7.1 U⋅L. In our perception, this assay has a large potential with respect to diagnoses of DNase I activity-related diseases and in drug screening. [ABSTRACT FROM AUTHOR]

Rheumatology laboratory tests are often inappropriately ordered in situations for which they are of low diagnostic utility. We surveyed pediatricians to investigate reasons for ordering these tests. The response rate was 15.3% (93/609). The most commonly ordered tests were antinuclear antibody (ANA), anti-double-stranded DNA (dsDNA) antibody, and rheumatoid factor (RF). Of the 89% (83/93) who ordered an ANA, 86% ordered it for correct/possibly correct reasons; of the 73% (68/93) who ordered RF, 80% did so for correct/possibly correct reasons; and among the 59% (54/92) who had ordered anti-dsDNA antibody, 34% ordered it for correct reasons. A positive relationship was seen between years since residency completion and correct use of ANA. However, positive associations were not seen between measures of pediatric rheumatology experience and correct use of other tests. Interventions are needed to improve pediatricians’ utilization of rheumatology tests. [ABSTRACT FROM AUTHOR]

Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material. [ABSTRACT FROM AUTHOR]

The differences in chromatographic behaviour of individual deoxynucleotides as well as small single-stranded and double-stranded DNA molecules have been examined for two resins from the Capto family: Capto Adhere and Capto Q ImpRes. Capto Adhere carries a multimodal ligand which combines strong anion with aromatic recognition, while Capto Q ImpRes is a strong anion exchanger with a chemically similar ligand, but without a phenyl group. The intrinsic differences between single- and double-stranded DNAs are related to charge, hydrophobicity, size and three-dimensional structure. These variations in biophysical properties have been utilized for comparative separations on these two resins. All deoxynucleotides and DNAs tested bound strongly to the chromatographic materials and could be eluted by a linear gradient of increasing NaCl concentration. Capto Q ImpRes provided a recognition for guanylate bases when samples of deoxynucleotides or poly(dG) were examined. This recognition was not observed for Capto Adhere. Another pronounced difference between the resins was observed in the inverted elution of ss- and dsDNA, where ssDNA eluted at 2.88 M NaCl on Capto Adhere, while on Capto Q ImpRes ssDNA eluted already at 1.47 M NaCl. This behaviour can be linked to the presence of the more hydrophobic phenyl group in Capto Adhere, leading to stronger retention of ssDNA molecules, which have a more hydrophobic character due to a higher degree of base exposure. [ABSTRACT FROM AUTHOR]

A novel label-free electrochemical impedance aptasensor based on a gold nanoparticles/double-stranded DNA-graphene (AuNPs/dsDNA-GO) nanocomposite modified glassy carbon electrode was presented for quantitative determination of thrombin. GO was covalently functionalized with dsDNA via a facile amidation process, and then AuNPs were electrodeposited onto the surface of dsDNA-GO. The morphology, conductivity and interaction of the as-prepared nanocomposites were characterized by scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy (EIS), Raman and Fourier transform infrared spectroscopy. The thrombin-binding aptamer (TBA) was conjugated to AuNPs via gold-thiol chemistry to construct electrochemical aptasensing platform, and the specific recognition between TBA and thrombin was monitored by EIS. Under optimum conditions, thrombin could be quantified in a wide range of 0.1–100 nM ( R 2 = 0.9960) with low detection limit of 0.06 nM (S/N = 3). [ABSTRACT FROM AUTHOR]

The rheology of lyotropic cholesteric liquid crystalline phase forming double-stranded DNA (dsDNA) stabilized single-wall carbon nanotube (SWCNT) dispersions was analyzed over a wide range of SWCNT concentrations. In the semidilute regime, supernatant dispersions of dsDNA-SWCNTs showed the characteristic rheological behavior of semidilute solutions of rods predicted by the Doi-Edwards theory. These cholesterogenic liquid crystal dispersions exhibited some of the key rheological behaviors associated with lyotropic liquid crystalline polymers (LLCPs) including a non-monotonic relationship in the viscosity and moduli versus concentration curves, and the empirical Cox-Merz not being obeyed. However, the dsDNA-SWCNT viscosity versus shear rate curves exhibited an inflection point instead of the three distinct regions associated with many LLCPs and nanocylinder dispersions. This could be related to orientation defects and inherent secondary interactions between the dsDNA and the SWCNTs. Linear viscoelasticity measurements also showed similarities to other lyotropic systems. [ABSTRACT FROM AUTHOR]

TIP49a and TIP49b, highly conserved proteins belonging to the AAA superfamily of DNA-dependent ATPases, participate in numerous cell processes, such as chromatin remodeling, regulation of gene transcription and mitotic cell division, maintenance of genome stability, and snoRNP biogenesis, as well as in the formation of active DNA-telomerase complexes. It has been shown that they are involved in complex networks of protein-protein interactions and, in spite of their structural similarity, sometimes perform opposite functions. Although these proteins exhibit a wide range of activities, the mechanisms of their actions are still poorly understood. In this work, ring-shaped heterohexameric TIP49a/b complexes containing in their central channel short fragments of double-stranded DNA (dsDNA, 20 base pairs of different GC composition) were obtained for the first time using the molecular docking technique, while methods of molecular dynamics in a periodic water box were applied to investigate the conformational dynamics of these proteins and the mechanisms of their helicase activity. It was found that (1) interaction of a DNA helix with positively charged protein loops inside the central channel of the ring-shaped hexameric complex caused unwinding of the helix; (2) the unwinding occurred only inside the hexameric ring, whereas the tails of the helix, which lie outside, retained the initial classical B-form conformation throughout the 50 ns of molecular dynamics; and (3) the presence of ATP in TIP49a/b complexes affected the dynamics and the final structure of dsDNA, causing partial breaking of complementary bonds in GC-poor DNA sequences. [ABSTRACT FROM AUTHOR]

Enhanced sequence-specific recognition of double-stranded DNA (dsDNA) was realized by using hybridization chain reaction (HCR) and positively charged gold nanoparticles ((+)AuNPs) dual signal amplification. To construct such a sensor, capture probe was initially assembled onto gold electrode surface. Upon addition of dsDNA, sandwiched DNA complex was formed between the capture probe and the detection probe, then another exposed part of the detection probe opened two alternating DNA hairpins (H 1 and H 2 ) in turn and initiated HCR to form a double-helix. Meantime, (+)AuNPs were electrostatically adsorbed onto such double-helix to amplify the electrochemical signal. Upon optimal conditions, the electronic signals of ferrocene (Fc) that modified on H 1 and H 2 increased linearly with increasing dsDNA concentration over the range from 15 pM to 1.0 nM, with a detection limit of 2.6 pM. Moreover, the proposed method showed good sequence specificity for dsDNA recognition. [ABSTRACT FROM AUTHOR]

In our earlier studies, we observed that when mice are treated with cyclophosphamide and fragmented exogenous dsDNA (18–30 h post cytostatic treatment), they develop a very characteristic set of symptoms and 80–90% of such animals succumb within 6–25 days. This was called “delayed death” phenomenon, and the gap between cyclophosphamide and DNA injections required for such phenotype to develop was termed “death window”. We established that mice succumbed to multi-organ failure, which was caused by systemic inflammation and sepsis. These processes unfolded along with accidental involution of lymphoid organs, which resulted from the failure of CD34 + hematopoietic stem cells to differentiate into lymphoid lineage progenitors. Here we compare SPF and non-SPF animals, and demonstrate that the major cause of systemic inflammation and sepsis observed upon such treatments is activation of an opportunistic infection. Mice of the same strain (CBA) housed under SPF conditions do not develop the characteristic symptoms, nor do they become moribund. Yet, regardless of the breeding conditions, upon synergistic action of cyclophosphamide and dsDNA, CD34 + hematopoietic stem cells consistently fail to give rise to lymphoid lineage progenitors. We demonstrate that this differentiation defect is reversible and that population of lymphoid progenitors is restored by day 29 after cyclophosphamide injection. [ABSTRACT FROM AUTHOR]

Previously, we reported our investigations of the interaction between a cyclic naphthalene diimide derivative (cNDI 1 ) and double stranded DNA (dsDNA) ( Bioorg. Med. Chem. 2014 , 22 , 2593). Here, we report the synthesis of the novel cNDI 2 , which has shorter linker chains than cNDI 1 . We performed comparative investigations of the interactions of both cNDI 1 and cNDI 2 with different types of dsDNA, including analysis of their thermodynamics and kinetics. Interactions between the cNDIs and calf thymus DNA (CT-DNA), poly[d(A-T)] 2 , or poly[d(G-C)] 2 were explored by physicochemical and biochemical methods, including UV–Vis spectroscopy, circular dichroism (CD) spectroscopy, stopped-flow kinetics, and a topoisomerase I assay. Upon addition of cNDIs to CT-DNA, the existence of an induced CD signal at approximately the wavelength of the naphthalene diimide chromophore and unwinding of the DNA duplex, as detected by the topoisomerase I assay, revealed that cNDIs bound to the DNA duplex. As indicated by the steric constraint in the formation of the complex, bis-threading intercalation was the more favorable binding mode. UV–Vis spectroscopic titration of the cNDIs with DNA duplexes showed affinities on the order of 10 5 –10 6 M −1 , with a stoichiometry of one cNDI molecule per four DNA base pairs. Thermodynamic parameters (Δ G , Δ H , and Δ S ) based on the van’t Hoff equation indicated that exothermic and entropy-dependent hydrophobic interactions played a major role in the reaction. Stopped-flow association and dissociation analysis showed that cNDI interactions with poly[d(G-C)] 2 were more stable and had a slower dissociation rate than their interactions with poly[d(A-T)] 2 and CT-DNA. Measurement of ionic strength indicated that electrostatic attraction is also an important component of the interaction between cNDIs and CT-DNA. Because of its longer linker chain, cNDI 1 showed higher binding selectivity, a more entropically favorable interaction, and much slower dissociation from dsDNA than cNDI 2 . [ABSTRACT FROM AUTHOR]

Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. [ABSTRACT FROM AUTHOR]

A new gene detection platform based on surface-enhanced Raman spectroscopy (SERS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system is developed, which is applied for virus gene detections. CRISPR/Cas12a activated by CRISPR ribonucleic acid (crRNA) can recognize and lock the complementary target double-stranded deoxyribonucleic acid (dsDNA), followed by the cis -cleavage on the dsDNA and the trans -cleavage on the nearby linker single-stranded DNA (ssDNA). Target-induced trans -ssDNA cleavage leads to a failure in an aggregation behavior of the designed SERS probe pair that was bridged by the linker ssDNA, which causes the SERS intensity reduced relative to the control trial that gives much stronger SERS due to the severe aggregation state of SERS probes caused by intact linker ssDNA. This sensing method achieves the highly sensitive detections of human papillomavirus (HPV) genes in serum and pseudovirus within 40 min. The detection sensitivity of this CRISPR/Cas-SERS platform can reach pM. Owing to the flexible designs of the DNA probe and crRNA, this CRISPR/Cas-SERS sensing system is a universal gene detection tool, which can be applied for the in-vitro diagnosis field and point-of-care testing. • A combination of CRISPR/Cas and SERS technology for virus double-stranded DNA detection. • High sensitivity of SERS for ultra-low concentration DNA. • It is un-amplification gene detection technology. • This design is versatile and universal for double-stranded DNA detection. [ABSTRACT FROM AUTHOR]

In this study a novel biosensor for determination of taxol is described. The interaction of taxol with salmon-sperm double-stranded DNA (ds-DNA) based on the decreasing of the oxidation signals of guanine and adenine bases was studied electrochemically with a pencil-graphite electrode (PGE) using a differential pulse voltammetry (DPV) method. The decreases in the intensity of the guanine and adenine oxidation signals after interaction with taxol were used as indicator signals for the sensitive determination of taxol. DPV exhibits a linear dynamic range of 2.0×10 −7 –1.0×10 −5 M for taxol with a detection limit of 8.0×10 −8 M. Finally, this modified electrode was used for determination of taxol in some real samples. [ABSTRACT FROM AUTHOR]

Background: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. Methods: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. Results: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. Conclusions: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction. [ABSTRACT FROM AUTHOR]

This research aims to provide new information about the mechanical behavior of double-stranded DNA (dsDNA). For this purpose, a series of extended atomic resolution molecular dynamics (MD) simulations of DNA dodecamer is performed. The MD calculations are carried out using Generalized Born solvent-accessible surface area method and Langevin dynamics. The stress-strain curves of DNA obtained under various pulling rates and pulling angles are analyzed, and the role of pulling angle and velocity in determining biomechanical properties of short dsDNA is discussed. The results illustrate that how much the behavior of DNA under action of tensile forces could be complicated. By means of at base pair level analyses of the molecule conformation during the stretching processes, the structural stability of the DNA molecule subjected to the angled pulling with different pulling rates and different pathways to the dsDNA rupture are studied. The structural stability of dsDNA can be dependent on the pulling velocity and pulling angle. Whereas the DNA stability can decrease significantly with the reduction of pulling velocity, stretching the DNA under different angles has different unpredictable effects on its structural stability. [ABSTRACT FROM AUTHOR]

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes–derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection. [ABSTRACT FROM AUTHOR]

The behavior of a mixture of short fragments of double-stranded DNA and a flexible-chain polymer is addressed. The role of the DNA double helix in the formation of the crystalline phase in the flexible-chain polymer's melt is examined. It is shown that the presence of DNA rigid fragments in melt results in increase in the melting temperature. A shift of the melting temperature of a perfect crystal with respect to the pure melt of polymer is calculated. [ABSTRACT FROM AUTHOR]

Abstract: Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au–S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350pM to 25nM, with a detection limit of 275pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches. [Copyright &y& Elsevier]

Phase behavior of short double-stranded DNA molecules dissolved in the water-containing polymer matrix is considered theoretically. Flexible-chain polymer molecules are assumed to be neutral. The phase behavior of DNA fragments is shown to be governed by the effective attraction, occurring due to polyvalent cations dissolved in water, and the osmotic pressure of the flexible-chain polymer. [ABSTRACT FROM AUTHOR]