DNA Releasing Research Articles

Alkylated Sulfonium Modification of Low Molecular Weight Polyethylenimine to Form Lipopolymers as Gene Vectors.

Hydrophobic modification of low molecular weight polyethylenimine (PEI) is an efficient method to form ideal gene-transfer carriers. Sulfonium-a combination of three different functional groups, was conjugated onto PEI 1.8k at a conjugation ratio of 1:0.1 to form a series of sulfonium PEI (SPs). These SPs were hydrophobically modified and characterized by Fourier transform infrared and HNMR. DNA-condensing abilities of SPs were tested with gel retardation experiment, and their cytotoxicity was evaluated via the MTT assay. The particle size and zeta potential of SP/DNA nanoparticles were measured and evaluated for cellular uptake and transfection ability on HepG2 cell line. The results showed that the sulfonium moiety was attached to PEI 1.8k with a high yield at a conjugation ratio of 1:0.1. SPs containing longer alkyl chains condensed DNA completely at an SP/DNA weight ratio of 2:1. The formed nanoparticle size was in the range of 168-265 nm, and the zeta potential was +16-45 mV. The IC50 values of SPs were 6.5-43.2 μg/mL. The cytotoxicity of SPs increased as the hydrophobic chain got longer. SP/DNA showed much stronger cellular uptakes than PEI 25k; however, pure SPs presented almost no gene transfection on cells. Heparin release experiment showed that SP's strong binding of DNA resulted in low release of DNA and thus hindered the gene transfection process. By mixing SP with PEI 1.8k, the mixture presented adjustable DNA binding and releasing. The mixture formed by 67% SP and 33% PEI 1.8k showed strong gene transfection. In conclusion, sulfonium is an effective linkage to carry hydrophobic groups to adjust cell compatibilities and gene transfection capabilities of PEI.

Schottky Heterojunction Realizes In Situ Vaccine‐Like Antitumor Efficacy and Microenvironment Remodeling Upon Near‐Infrared Laser Response in Cold Tumors

AbstractCold tumor is one of the most refractory tumors due to its low immunogenicity and absence of T cell infiltration. The immunotherapeutic effect of near‐infrared (NIR) responsive nanomaterials on tumors is far from satisfactory. Herein, ultrasmall γ‐MnO2 nanodots are anchored on the intrinsic metallic Ti3C2(OH)2, modified with bovine serum albumin, to realize a Schottky heterojunction (labeled as TC‐MnO2@BSA), which can be utilized to reshape the cold tumor microenvironment (TME) through in situ vaccine‐like antitumor effect. The Schottky heterojunction endows TC‐MnO2@BSA with improved photothermal conversion and reactive oxygen species (ROS) generation. Excess ROS and heat lead to tumor immunogenic death (ICD) and abundant damaged double‐strain DNA releasing into TME, coordinated with TC‐MnO2@BSA‐derived Mn2+, magnifying the cGAS‐STING signaling pathway, eventually promoting antigen presentation of dendritic cells and infiltration of T cells. Such a NIR‐activated nanovaccine can achieve complete ablation of tumors while robust activating systemic antitumor immune response. Furthermore, it inhibits the growth of abscopal tumors through dramatically “heating” their cold TME. This work introduces a universal strategy to magnify the photothermal and immune adjuvant effect through the gain of Schottky heterostructure, as a novel paradigm to construct the multifunctional in situ nanovaccine.

Novel diamide resistance-linked mutation in Korean Spodoptera exigua and a LAMP assay based on a mutation-associated intronic InDel

Recently, resistance to diamide insecticides has been reported in various lepidopteran pests, including Spodoptera exigua. Six field populations and a local population were tested, and results revealed high levels of diamide resistance. We selected a diamide-resistant strain, showing LC50 values 28,950- and 135,286-fold higher than those of a susceptible strain against chlorantraniliprole and flubendiamide, respectively. However, G4946E mutation in the ryanodine receptor, one of the well-known diamide resistance mechanisms, was not found in the tested resistant field populations. Instead, through genome sequencing, we found an I4790M mutation and some InDels, particularly a 29-bp insertion in the neighboring intron that was associated with this resistant allele. By using this distinct region, resistant allele diagnostic primers were designed and applied in a lamp loop-mediated isothermal amplification (LAMP) assay and general PCR. The applicable temperature of the LAMP assay was from 63 to 65 °C for 2 h with four primers. Addition of a loop primer further increased the amplification efficiency. LAMP was feasible with a broad range of DNA concentrations, with a minimum detectable concentration of 100 pg. A DNA releasing method comprising 5 min of incubation at 95 °C allowed DNA detection from some larva and adult samples. The combination of this DNA releasing technique and LAMP resulted in a rapid (up to 100 min) and accurate diagnostic strategy that could be applied to monitor and manage diamide resistance in S. exigua.

Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP.

Simple SummaryMythimna loreyi is a serious pest of grain crops and reduces yields in maize plantations. M. loreyi is a native species in East Asia for a long time. However, this species has recently emerged as a migration (or invasive from other Asian countries) pest of some cereal crops in Korea. Little is known about its basic biology, ecology, and it is difficult to identify the morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Species diagnosis methods for invasive pests have been developed and utilized for this reason. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198) and process PCR and sequencing to compare the degree of homology. However, this method requires a lot of time and effort. In this study, we developed the LAMP (loop-mediated isothermal amplification) assay for rapid, simple, and effective species diagnosis. By analyzing the mitochondrial (mt) genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. A broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process, and only some pieces of tissue from larvae and adult samples were needed. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly used in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.The Mythimna loreyi (Duponchel) is one of the well-known invasive noctuid pests in Africa, Australia, and many Asian countries. However, it is difficult to identify the invasive and morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198), which requires a lot of time and effort, such as DNA extraction, PCR, electrophoresis, and sequencing. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mitochondrial genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific, in general, and multiplex PCR and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 °C for 60 min with four LAMP primers, though additional loop primers, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process. Only some pieces of tissue of larvae and adult samples were needed to extract DNA. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly applied in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. Totally 5 screening elements (P–35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P–35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.

Sensing Interfaces for Bioelectrocatalytic Stimulated Release

Signal-stimulated (bio)molecular release has been extensively studied and reported in numerous research papers, reviews and books,1 being motivated by various biomedical and biotechnological applications. Among many other molecular, biomolecular and nano-size species, DNA molecules have been studied for the signal-controlled release, being highly important for gene delivery therapy, biosensors, biochips, unconventional biocomputing, and for many other applications. Various signals triggering DNA release have been used including application of altering electromagnetic field, photochemical capture and release of DNA, thermally stimulated release of DNA, etc. Electrochemically triggered DNA release is particularly attractive due to its simple realization and versatility. Specifically, these developed systems are designed for different release processes triggered by various signals (electrical, biomolecular, illumination, etc.), thus representing a general interfacial platform for controlled release of different biomolecules and nano-size species.2 Herein, we report on the DNA releasing system controlled by local/interfacial pH changes triggered by a very small potential applied to reduce oxygen bioelectrocatalytically with the help of immobilized bilirubin oxidase (Box). Notably, the DNA release was only a convenient model, while the developed approach can be adapted to the electrically stimulated release of any negatively charged (bio)molecules or nano-species.3 1. Li, X.; Jasti, B. R. Design of Controlled Release Drug Delivery Systems, McGraw-Hill Education, New York, 2005.2. Masi, M.; Bollella, P.; Katz, E. Electroanalysis 2019, 31, in press, published on-line (DOI: 10.1002/elan.201900377).3. Masi, M.; Bollella, P.; Katz, E. ACS Applied Materials and Interfaces 2019, Submitted.

Nicotine Enhances Staphylococcus epidermidis Biofilm Formation by Altering the Bacterial Autolysis, Extracellular DNA Releasing, and Polysaccharide Intercellular Adhesin Production.

Staphylococcus epidermidis is a common bacterial colonizer of human skin and mucous membranes, yet it has emerged as an important nosocomial pathogen largely due to its ability to form biofilms. Tobacco smoke has been demonstrated as a contributor to various infection diseases by improving the biofilm formation of multiple bacterial species; however, the association between tobacco smoke and S. epidermidis biofilm is still unclear. In this study, we tested the effect of nicotine, one of the most active components of tobacco, on S. epidermidis biofilm formation, and we studied the underlying mechanisms. Our results showed that nicotine promoted the biofilm formation of S. epidermidis 1457 strain (SE1457) and enhanced its initial attachment to a polyethylene surface as well as polysaccharide intercellular adhesin (PIA) production. In addition, an increased extracellular DNA release and a higher autolysis rate of SE1457 was detected after nicotine treatment, which was consistent with the increased ratio of dead cells in nicotine-treated SE1457 biofilm observed with confocal laser-scanning microscopy. Furthermore, the effect of nicotine on several autolysis-related and biofilm-related gene knockout mutants of SE1457 was tested. It showed that in ΔsaeRS, ΔlytSR, and ΔsceD, nicotine induced increase in biofilm formation was similar to that in SE1457; but in ΔarlRS, ΔatlE, and ΔicaC, the effect was obviously impaired. Consistently, the increase of the bacterial autolysis rate in ΔarlRS and ΔatlE induced by nicotine was not as significant as that in SE1457. Meanwhile, the growth inhibition of nicotine on SE1457 was observed, and it was much less on ΔarlRS and restored by the arlRS complementation. The arlRS transcription in SE1457 was inhibited by nicotine during cultivation as indicated by a promoter reporter assay using green fluoresent protein. Taken together, our study indicates that nicotine improves S. epidermidis biofilm formation by promoting its initial attachment and intercellular accumulation; the arlRS, atlE, and ica genes mediating bacterial autolysis and PIA production play an important role in this process.

New method to preserve the original proportion and integrity of urinary cell-free DNA.

Due to high nuclease activity and complex contents in urine, urinary cell-free DNA (ucfDNA) was prone to degrade. So, we developed standardized urine collection tube (UCT) to prevent ucfDNA degradation and simultaneously maintain urinary cells in their original form during the sample collection process, ensuring stabilization of the original proportion and integrity of ucfDNA. Urine samples were collected from bladder cancer patients and divided into 10-mL normal tubes and 10-mL UCTs, respectively, and kept at ambient temperature. Urine supernatant was separated by centrifuging, and ucfDNA was extracted. Then ucfDNA was quantified by quantitative real-time polymerase chain reaction. UcfDNA fragments distribution was analyzed by Agilent 2200, and the frequency of specific mutations of urinary system disease was detected by next-generation sequencing method. Urine collected into UCTs showed no statistically significant changes in their original proportion and integrity of ucfDNA up to 7days at ambient temperature and also ucfDNA fragments were maintained well. Conversely, urine collected into normal tubes was observed an obviously decline in their original proportion of ucfDNA and ucfDNA fragments changed greatly. The △% of allele fraction (AF) for specific genes of ucfDNA from UCTs was lower than from normal tubes by 3.7-fold. Using UCTs, they can maximally keep the original proportion and integrity of ucfDNA and stabilize urinary cells and minimize the background noise caused by urinary cellular DNA releasing, it will be help to open the door of next-generation noninvasive liquid biopsy applications utilizing urine.

Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.

To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.

{Ru(CO)x}-core terpyridine complexes: Lysozyme binding affinity, DNA and photoinduced carbon monoxide releasing properties

Reaction of 4′-(2-pyridyl)-2,2′:6′,2″-terpyridine (LPY) and 4′-(4-phenylmorpholine)-2,2′:6′,2″-terpyridine (Lmorph) with {[RuCl2(CO)3]}2 in methanol affords [RuCl2(LPY-κ2N1N2)(CO)2] (1) and a mixture of [RuCl2(Lmoph-κ2N1N2)(CO)2]/[RuCl2(Lmorph-κ3N1N2N3)(CO)] (2), respectively. Their photoactivatable CO releasing properties are investigated upon the exposure to light source at 365 nm. One CO molecule is released from 1 at the excitation wavelength 365 nm, while the ligand changes its bidentate mode into the meridional tridentate one. The illumination profile and the influence of the uncoordinated pyridine arm on CO release are examined by solution 1H and 13C NMR spectroscopy. The electronic transitions are studied by TDDFT. The DNA and hen white egg lysozyme binding affinity of the complexes are studied by UV/Vis. and electrospray ionization mass spectrometry. Stable lysozyme complexes, capable of photo induce CO, are formed via the loss of the labile chloride ligands or terpyridine moiety.

Antimicrobial activity of Amazon Astrocaryum aculeatum extracts and its association to oxidative metabolism

Several compounds present in fruits as polyphenols are able to kill or inhibit the growth of microorganisms. These proprieties are relevant mainly in tropical areas, as Amazonian region where infectious are highly prevalent. Therefore, this study investigated the antimicrobial activity of tucumã Amazonian fruit against 37 microorganisms. The potential role of oxidative metabolism imbalance was also studied as causal mechanism of antimicrobial activity. The results showed antibacterial effect of pulp and peel tucumã hydro-alcoholic extracts on three Gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Listeria monocytogenes) and antifungal effect against Candida albicans. The antimicrobial contribution of main chemical compounds (quercetin, rutin, β-carotene and gallic, caffeic and chlorogenic acids) found in tucumã extracts was also investigated showing an inhibitory effect depending of the organism mainly by quercetin in bacteria and rutin in C. albicans. Analysis of kinetic of DNA releasing in extracellular medium by fluorescence using DNA Pico Green assay® and reactive oxygen species production (ROS) showed potential oxidative imbalance contribution on tucumã inhibitory effect. In B. cereus and C. albicans this effect was clear since after 24h the ROS levels were higher when compared to negative control group. In conclusion, tucumã extracts present antimicrobial activity to four microorganisms that have large problems of drug resistance, and the possible mechanism of action of this Amazon fruit is related to REDOX imbalance.

Engineering Buffering and Hydrolytic or Photolabile Charge Shifting in a Polycarboxybetaine Ester Gene Delivery Platform

Polycarboxybetaine esters (PCB-esters) can condense plasmid DNA into nanosized polyplexes for highly effective gene delivery with low toxicity. The design and characterization of tertiary CB-ester monomers and PCB-ester polymers are presented here to study the effects of molecular variation on functions important to nonviral gene transfer. Both buffering capacity and charge-shifting behavior can be tuned by modifying the distance between the charged groups and the ester size or type. A carbon spacer length (CSL) of one was found to bring the pKa of the tertiary amine into the optimal range for proton buffering. Ester hydrolytic degradation switches this polymer from cationic (DNA binding) to zwitterionic (DNA releasing) form while conferring nontoxicity. To allow rapid and externally controlled degradation, the effect of this charge-switching behavior on DNA release from polyplexes was directly studied with a novel photolabile PCB-nitrobenzyl ester (PCB-NBE). Photoinitiated ester degradation precipitated the rapid release of 72±5% of complexed DNA from PCB-NBE polyplexes. These insights reveal the key parameters important for the PCB-ester platform and the significance of charge switching to an effective and nontoxic nonviral gene delivery platform.

Plant bio-transformable HMG-CoA reductase gene loaded calcium phosphate nanoparticle: in vitro characterization and stability study.

Encapsulation of plasmid DNA in nanoparticle is expected to enhance the stability of DNA, reproducibility and frequency of the genetic transformation in plants. Here we report the formulation of HMG Co-A reductase gene loaded calcium phosphate nanoparticles (Cap nanoparticles) and their in-vitro, in-vivo characterization. The developed Cap nanoparticles were characterized by DSC, FT-IR, and XRD. Developed Cap nanoparticles were spherical in shape having the particle size and zeta potential in the range of 10.86±0.09nm to 33.42±0.18nm and -25.5±0.07mV to -31.7±0.07mV (for Cap-I to Cap-IV). DNA releasing in acidic media showed, initially slow release followed by fast release with a maximum release of Cap-I (95.77±1.39%) > Cap-II (87.32±2.07%) > Cap-III (76.54±2.01%) > Cap-IV (72.93±1.75%) over 60min. Cap nanoparticles were quite stable at storage condition of 40±0.5°C/75±5%RH, 25±0.5°C/60±RH, 4±0.5°C/ambient humidity and the integrity of pDNA encapsulated was confirmed by gel electrophoresis. Compared to wild type C. intybus, transformation efficiency and enhanced biosynthesis of esculin with the DNA nanoparticles in C. intybus were about 10% and 71%, respectively. Antioxidant activity capacity of the biotransformed plants was significantly higher than the normal plant due to high accumulation of esculin.

Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan

Previously, we had reported improving transfection efficiency of the chitosan-plasmid DNA (CS/pDNA)complex via enhancing intracellular unpacking of the exogene by the utilization of phosphorylatable short peptide conjugated chitosan (pSP-CS). In this article, we addressed a novel strategy of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) modification for further optimization of the transfection efficiency. NNS, consisting of “PKKRKVREEAIKFSEEQRFRR”, contained a SV40 nucleus localization signal and a potentially phosphorylatable serine residue. The short peptide could be selectively phosphorylated in the nucleus in various mammalian cells. This phosphorylatable NNS (pNNS) was conjugated to chitosan and combined with Cy3 fluorescence labeled plasmid DNA to form a pNNS-CS/pDNA complex. In vitro phosphorylation and DNA releasing assays verified that pNNS could be effectively and selectively phosphorylated by nucleic lysate, hence promoting pDNA unpacking from the complex. Thereafter, C2C12 myoblast cells were transfected. Nuclear localization of the pDNA was represented by the fluorescence in the nucleus and transfection efficiency was determined by the expression of the luciferase reporter gene, which is carried by the plasmid DNA. The results revealed that, compared with lipofactamine2000 and the previously reported pSP-CS, pNNS-CS could transport more pDNA into the nucleus and intensively augment luciferase reporter gene expression. In conclusion, nucleus localization and unpacking from the delivery vector are both critical factors in influencing exogene expression, and pNNS modification is valuable in improving transfection efficacy of the chitosan.

Phosphorylatable short peptide conjugation for facilitating transfection efficacy of CS/DNA complex

Previously, we had demonstrated that enhancing intracellular unpacking of exogene from its chitosan carrier by promoting chitosan degradation could markedly improve transfection efficiency of the CS/DNA complex. In this article we addressed a novel strategy of phosphorylatable short peptide modification for further facilitating intracellular DNA unpacking and optimizing transfection efficiency of the CS/DNA complex. A short peptide (SP) with the amino acid composition of “LLLRRRDNEY*FY*VRRLL” containing two potentially phosphorylatable tyrosine residues was synthesized. The short peptide could be phosphorylated by constitutively expressed cytoplasmic protein kinase Jak2. The SP was conjugated to chitosan and combined with GFP/luciferase reporter gene plasmid DNA to form SP-CS/DNA complex. In vitro phosphorylation and DNA releasing assays verified that mammalian cell lysate could effectively phosphorylate SP and hence promote plasmid DNA unpacking from the SP-CS carrier. Thereafter, C2C12 myoblast cells were transfected by SP-CS/DNA and the transfection efficiency was presented by the expression of GFP and luciferase reporters. Further more, multiple cell lines were transfected by SP-CS/DNA complexes loading luciferase reporter gene. Results revealed that, compared with CS, SP-CS could intensively augment the transfection efficiency to the level of near lipofectamine 2000.

Abstract 3465: DNA adducts of tobacco-specific nitrosamines in human pancreatic tissue resulting from pyridylhydroxybutylation and pyridyloxobutylation

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Whereas cigarette smoking is a well established important risk factor for pancreatic cancer, the contribution of smokeless tobacco consumption is still a matter of debate. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known compound present in tobacco and tobacco smoke which induces pancreatic tumors in animal models. In rats, the major NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), proved to be an even more potent pancreatic carcinogen. NNK and NNAL have been detected in pancreatic juice. However, activation of NNK and NNAL leading to pyridyloxobutyl (POB) and pyridylhydroxybutyl (PHB) DNA adducts, respectively, has not been proven in human pancreas. Determination of POB DNA adducts was achieved by acid hydrolysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) from DNA after addition of [3,3,4,4-D4]-HPB as internal standard, derivatization with pentafluorobenzoyl chloride (PFBC) and quantification by gas chromatography/high resolution mass spectrometry with negative chemical ionization according to Heppel et al. (Anal Bioanal Chem 393:1525-30, 2009). POB DNA adducts could be easily detected in 8 of 11 pancreatic samples of sudden death victims from Germany at 2.95±1.08 fmol/µg DNA (mean±standard error; values below detection limit set at zero). Using [3,3,4,4-D4]-diol as internal standard, this analytical method also revealed the presence of PHB adducts in pancreatic DNA releasing 4-hydroxy-1-(3-pyridyl)-1-butanol (diol). However, because of massive peak broadening the PFBC ester of diol could not be quantified reliably. To improve the chromatographic resolution, the PFBC ester of diol was further derivatized according to Kao and Giese (Chem Res Toxicol 18:70-5, 2005) with pivalic anhydride to cap the secondary hydroxy group. The resulting tert-butyl esters of the PFBC esters of diol and [3,3,4,4-D4]-diol show excellent performance in capillary gas chromatography and allow quantification by single ion monitoring at the molecular masses of m/z 445.13 and m/z 449.16, respectively. In summary, the presence of POB and PHB DNA adducts in human pancreatic tissue has been clearly demonstrated for the first time. Further studies will show the specificity of these pancreatic DNA adducts for smoking and smokeless tobacco consumption and the correlation with other tobacco-specific biomarkers. Supported by a grant of the Institute for Science and Health, St. Louis, MO. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3465.

Development of thiol-responsive amide bond cleavage device and its application for peptide nucleic acid-based DNA releasing system

To develop a thiol-responsive DNA-releasing system, a thiol-responsive amino acid capable of inducing an amide bond cleavage in the presence of a thiol was developed. It was successfully combined with peptide nucleic acid (PNA), and thiol-induced release of DNA from the thiol-responsive PNA/DNA complex was observed.

Is there ferroelectricity in DNA?

This paper reports on a study of the dielectric properties of DNA (sodium salt from calf thymus, SIGMA) under heating and cooling of a sample within the temperature interval 20–60°C, which was conducted in air, in a humid environment, and in vacuum. It is shown that the data obtained can be accounted for by the sample conductivity increasing as a result of DNA releasing water into a separate phase with increasing temperature and the formation of a double electric layer at the DNA interface with metallic electrodes, without invoking the idea of ferroelectricity present in DNA.

Plasmid adsorption to anion‐exchange matrices: Comments on plasmid recovery

A number of anion-exchange adsorbents were constructed, employing nonporous silica fibers, and examined with the aim of describing factors that influence desorption and recovery of plasmid DNA (pDNA). The fibers were provided with ligands via adsorption of the polymeric amines poly(ethyleneimine) or chitosan, or via graft-polymerization of primary, tertiary, or quaternary amine monomers to vinyl-silanized fibers. Several adsorbents showed an almost irreversible plasmid binding. It was suggested that important factors affecting the DNA releasing ability are (i) type of amine ligand used (primary amines bind plasmids the strongest), (ii) the structure of the nucleic acid (supercoiled pDNA may bind stronger than linear genomic DNA), (iii) shift of ligand pK(a) (due to the proximity of highly charged pDNA), and (iv) the solid support itself (steric factors may lead to kinetically stable complexes). The last factor was derived from several comparisons between support-bound ligand and free soluble ligand. It was thus observed that polyelectrolyte complexes associated with a surface were much more difficult to dissociate than the equivalent soluble complexes.

Intramuscular delivery of DNA releasing microspheres: Microsphere properties and transgene expression

Plasmid-loaded microspheres can provide localized and sustained release into the target tissue, and thus have the potential to enhance the efficiency of naked DNA at promoting transgene expression. In this report, microsphere design parameters are investigated by correlating the extent and duration of transgene expression intramuscularly to the polymer molecular weight and the mass of DNA delivered. Plasmid DNA was incorporated into poly (lactide- co-glycolide) microspheres using a cryogenic double emulsion process, and microspheres were injected intramuscularly. Bolus injection of naked plasmid was used for control, which exhibited transfection of muscle cells with transgene expression that gradually decreased over time. Microspheres fabricated from low molecular weight polymer had expression levels that increased from day 1 to day 92, which subsequently decreased through day 174. Decreasing the microsphere mass delivered resulted in steady expression during the same time. However, microspheres fabricated with high molecular weight polymer had expression for only 14 days. Intramuscular injection resulted in a foreign body response to the microspheres, and these infiltrating cells adjacent were primarily transfected. This understanding of microsphere properties that determine transgene expression and the distribution of transfected cells may facilitate their application to fields such as tissue engineering or DNA vaccines.