Test performance and acceptability of self- versus provider-collected swabs for high-risk HPV DNA testing in female-to-male trans masculine patients

PubMed Central

Deutsch, Madeline B.; Peitzmeier, Sarah M.; White Hughto, Jaclyn M.; Cavanaugh, Timothy P.; Pardee, Dana J.; McLean, Sarah A.; Panther, Lori A.; Gelman, Marcy; Mimiaga, Matthew J.; Potter, Jennifer E.

2018-01-01

Background High-risk human papillomavirus (hrHPV) causes virtually all cervical cancers. Trans masculine (TM) people (those assigned female at birth who identify with a gender other than female) have low uptake of conventional cervical cancer screening. Self-collected hrHPV DNA testing has high levels of acceptability among cisgender (non-transgender) females and may support increased cervical cancer screening uptake in TM individuals. Objective To assess the test performance and acceptability of self-collected vaginal specimens in comparison to provider-collected cervical swabs for hrHPV DNA detection in TM individuals ages 21–64 years. Methods Between March 2015-September 2016, 150 TM participants with a cervix (mean age = 27.5 years; SD = 5.7) completed a one-time study visit comprised of a self-report survey, self-collected vaginal HPV DNA swab, clinician-administered cervical HPV swab, and brief interview on acceptability of clinical procedures. Participants were randomized to complete either self- or provider-collection first to minimize ordering effects. Self- and provider-collected samples were tested for 13 hrHPV DNA types using a DNA Hybridization Assay. The primary outcome variable was the concordance (kappa statistic) and performance (sensitivity, specificity) of self-collected vaginal HPV DNA specimens versus provider-collected cervical HPV swabs as the gold standard. Results Of the 131 participants completing both the self- and provider-collected HPV tests, 21 cases of hrHPV were detected by the provider cervical swab (gold standard; 16.0% hrHPV prevalence); 15 of these cases were accurately detected by the self-collected vaginal swab (71.4% concordance) (Kappa = 0.75, 95% Confidence Interval [CI]: 0.59, 0.92; p<0.001). Compared to the provider-collected cervical hrHPV DNA sample (gold standard), the self-collected vaginal hrHPV DNA test demonstrated a sensitivity of 71.4% (95% CI: 0.52, 0.91; p = 0.0495) and specificity of 98.2% (95% CI: 0.96, 1.00; p<0.0001). Over 90% of participants endorsed a preference for the self-collected vaginal swab over provider-collected cervical swab. Conclusion Self-collected vaginal swabs are highly acceptable to TM as a means to test for hrHPV DNA. Test performance of this self-collection method for hrHPV detection in TM is consistent with previous studies in cisgender females. Self-collected vaginal swab testing for hrHPV DNA represents a reasonable and patient-centered strategy for primary cervical cancer screening in TM patients unwilling to undergo provider collection of specimens via speculum exam. PMID:29538411

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the Terminal Ileum of Fistulated Göttingen Minipigs

PubMed Central

Lick, Sonja; Drescher, Karsten; Heller, Knut J.

2001-01-01

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 106 to 107 per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth. PMID:11526016

A DNA Nanotube-Peptide biocomplex for mRNA Detection and Its Application in Cancer Diagnosis and Targeted Therapy.

PubMed

Ji, Xiaoting; Lv, Haoyuan; Guo, Jiayi; Ding, Caifeng; Luo, Xiliang

2018-04-25

A biocomplex of DNA nanotube-peptide, consisting of six concatenated DNA strands, three lock DNA strands and a cell-penetrating peptide was developed herein. The barrel structured DNA nanotube-peptide was successfully applied as a co-drug delivery system for targeting cancer therapy. The mucin 1 proteins (MUC-1) aptamer which is part of DNA nanotube can specially recognize MUC-1 protein on the surface of MCF-7 cells. Cyclo (Arg-Gly-Asp-D-phe-Lys) (cRGD), as a cell-penetrating peptide, facilitates recruitment and uptake of targeting drugs by binding to integrin receptors (αvβ3) of cytomembrane surface. Anti-cancer drug doxorubicin (DOX) and paclitaxel (PTX) were loaded into the capsulated DNA nanotube-peptide (CDNP), which was used as co-drug cargo models. The as-prepared biocomplex can be utilized not only to deliver drug but also to achieve the anticancer effect in vivo. The experimental results suggested that the treatment efficacy of co-drug delivery platform (CDNP/DOX/PTX) was better than single-drug delivery platform (CDNP/DOX or CDNP/PTX). This system that was composed by DNA strands and peptide has good biocompatibility and biodegradability. Furthermore, the system can readily achieve detection of target mRNA of MCF-7 cell in vitro. The detection limits of mRNA are 9.7×10-8 M and 1.8×10-8 M by using CDNP/DOX and CDNP/PTX-FITC as a probe, respectively. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

PubMed

Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

2016-01-01

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

PubMed

Schaffert, David H; Okholm, Anders H; Sørensen, Rasmus S; Nielsen, Jesper S; Tørring, Thomas; Rosen, Christian B; Kodal, Anne Louise B; Mortensen, Michael R; Gothelf, Kurt V; Kjems, Jørgen

2016-05-01

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

PubMed

Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A

2015-07-08

Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis

NASA Astrophysics Data System (ADS)

Levin, Galit; Shirvan, Anat; Grimberg, Hagit; Reshef, Ayelet; Yogev-Falach, Merav; Cohen, Avi; Ziv, Ilan

2009-09-01

Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N'-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

PubMed Central

Maruyama, Fumito; Kenzaka, Takehiko; Yamaguchi, Nobuyasu; Tani, Katsuji; Nasu, Masao

2005-01-01

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria. PMID:16332770

DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

NASA Astrophysics Data System (ADS)

Franch, Oskar; Iacovelli, Federico; Falconi, Mattia; Juul, Sissel; Ottaviani, Alessio; Benvenuti, Claudia; Biocca, Silvia; Ho, Yi-Ping; Knudsen, Birgitta R.; Desideri, Alessandro

2016-07-01

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01806h

DNA Uptake by Transformable Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lacks, Sanford A.

1999-03-31

The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

DNA UPTAKE BY TRANSFORMABLE BACTERIA

DOE Office of Scientific and Technical Information (OSTI.GOV)

LACKS,S.A.

1999-09-07

The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

Prokaryotic Information Games: How and When to Take up and Secrete DNA.

PubMed

Stingl, Kerstin; Koraimann, Günther

2017-01-01

Besides transduction via bacteriophages natural transformation and bacterial conjugation are the most important mechanisms driving bacterial evolution and horizontal gene spread. Conjugation systems have evolved in eubacteria and archaea. In Gram-positive and Gram-negative bacteria, cell-to-cell DNA transport is typically facilitated by a type IV secretion system (T4SS). T4SSs also mediate uptake of free DNA in Helicobacter pylori, while most transformable bacteria use a type II secretion/type IV pilus system. In this chapter, we focus on how and when bacteria "decide" that such a DNA transport apparatus is to be expressed and assembled in a cell that becomes competent. Development of DNA uptake competence and DNA transfer competence is driven by a variety of stimuli and often involves intricate regulatory networks leading to dramatic changes in gene expression patterns and bacterial physiology. In both cases, genetically homogeneous populations generate a distinct subpopulation that is competent for DNA uptake or DNA transfer or might uniformly switch into competent state. Phenotypic conversion from one state to the other can rely on bistable genetic networks that are activated stochastically with the integration of external signaling molecules. In addition, we discuss principles of DNA uptake processes in naturally transformable bacteria and intend to understand the exceptional use of a T4SS for DNA import in the gastric pathogen H. pylori. Realizing the events that trigger developmental transformation into competence within a bacterial population will eventually help to create novel and effective therapies against the transmission of antibiotic resistances among pathogens.

Biomarkers of human exposure to pesticides.

PubMed Central

Anwar, W A

1997-01-01

For centuries, several hundred pesticides have been used to control insects. These pesticides differ greatly in their mode of action, uptake by the body, metabolism, elimination from the body, and toxicity to humans. Potential exposure from the environment can be estimated by environmental monitoring. Actual exposure (uptake) is measured by the biological monitoring of human tissues and body fluids. Biomarkers are used to detect the effects of pesticides before adverse clinical health effects occur. Pesticides and their metabolites are measured in biological samples, serum, fat, urine, blood, or breast milk by the usual analytical techniques. Biochemical responses to environmental chemicals provide a measure of toxic effect. A widely used biochemical biomarker, cholinesterase depression, measures exposure to organophosphorus insecticides. Techniques that measure DNA damage (e.g., detection of DNA adducts) provide a powerful tool in measuring environmental effects. Adducts to hemoglobin have been detected with several pesticides. Determination of chromosomal aberration rates in cultured lymphocytes is an established method of monitoring populations occupationally or environmentally exposed to known or suspected mutagenic-carcinogenic agents. There are several studies on the cytogenetic effects of work with pesticide formulations. The majority of these studies report increases in the frequency of chromosomal aberrations and/or sister chromatid exchanges among the exposed workers. Biomarkers will have a major impact on the study of environmental risk factors. The basic aim of scientists exploring these issues is to determine the nature and consequences of genetic change or variation, with the ultimate purpose of predicting or preventing disease. PMID:9255564

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, R.M.M.; Martinez, J.R.

The uptake and efflux of the isotopic tracer /sup 45/Ca were compared in dispersed submandibular acini of both control rats and rats treated with seven daily doses of reserpine (0.5 mg/kg, i.p.). Tracer uptake occurred in a time-dependent manner in both types of acini and reached 8.4 +/- 0.2 and 8.0 +/- 0.2 pmol/mg protein, respectively, in acini from control and treated animals after 60 min of incubation. Uptake of tracer was 2.35 nmol/mg DNA in control cells and 4 nmol/mg DNA in cells from treated rats at 60 min. /sup 45/Ca uptake (per mg protein) was enhanced in controlmore » acini 48% by 20 mumol/L epinephrine; 38% by 50 mumol/L carbachol; and 23% by 10 mumol/L isoproterenol. A similar order of potency was observed when uptake was expressed per mg DNA. In acini from reserpine-treated rats, /sup 45/Ca uptake (per mg protein) was increased 53% by epinephrine, 39% by isoproterenol, and only 8% by carbachol. The same enhanced effect of isoproterenol and lack of effect of carbachol were observed when uptake was calculated per mg DNA. In the absence of secretagogue, efflux of /sup 45/Ca from tracer-pre-loaded acini was larger in acini from reserpine-treated rats (53%) than in control acini (36%). Whether expressed in terms of mg protein or mg DNA, this efflux was increased in control acini 35% by epinephrine, from 25 to 28% by isoproterenol, and 17% by carbachol. In acini of reserpine-treated rats, epinephrine increased /sup 45/Ca efflux 20%, isoproterenol from 25 to 28%, and carbachol from 14 to 15%.« less

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

PubMed

Ziraksaz, Zarrintaj; Nomani, Alireza; Ruponen, Marika; Soleimani, Masoud; Tabbakhian, Majid; Haririan, Ismaeil

2013-01-23

Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression. Copyright © 2012 Elsevier B.V. All rights reserved.

Occurrence of H2-Uptake Hydrogenases in Bradyrhizobium sp. (Lupinus) and Their Expression in Nodules of Lupinus spp. and Ornithopus compressus1

PubMed Central

Murillo, Jesús; Villa, Ana; Chamber, Manuel; Ruiz-Argüeso, Tomás

1989-01-01

Fifty-four strains of Bradyrhizobium sp. (Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H2 uptake by bacteroids and the relative efficiency of N2 fixation (RE = 1 - [H2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup+ strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. Images Figure 1 Figure 2 PMID:16666550

Investigation of the electrostatic and hydration properties of DNA minor groove-binding by a heterocyclic diamidine by osmotic pressure.

PubMed

Erlitzki, Noa; Huang, Kenneth; Xhani, Suela; Farahat, Abdelbasset A; Kumar, Arvind; Boykin, David W; Poon, Gregory M K

2017-12-01

Previous investigations of sequence-specific DNA binding by model minor groove-binding compounds showed that the ligand/DNA complex was destabilized in the presence of compatible co-solutes. Inhibition was interpreted in terms of osmotic stress theory as the uptake of significant numbers of excess water molecules from bulk solvent upon complex formation. Here, we interrogated the AT-specific DNA complex formed with the symmetric heterocyclic diamidine DB1976 as a model for minor groove DNA recognition using both ionic (NaCl) and non-ionic cosolutes (ethylene glycol, glycine betaine, maltose, nicotinamide, urea). While the non-ionic cosolutes all destabilized the ligand/DNA complex, their quantitative effects were heterogeneous in a cosolute- and salt-dependent manner. Perturbation with NaCl in the absence of non-ionic cosolute showed that preferential hydration water was released upon formation of the DB1976/DNA complex. As salt probes counter-ion release from charged groups such as the DNA backbone, we propose that the preferential hydration uptake in DB1976/DNA binding observed in the presence of osmolytes reflects the exchange of preferentially bound cosolute with hydration water in the environs of the bound DNA, rather than a net uptake of hydration waters by the complex. Copyright © 2017 Elsevier B.V. All rights reserved.

Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

PubMed Central

Ambur, Ole Herman; Frye, Stephan A.; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

2012-01-01

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID:22768309

Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules.

PubMed

Olsen, Chris M; Shikiya, Ronald; Ganugula, Rajkumar; Reiling-Steffensmeier, Calliste; Khutsishvili, Irine; Johnson, Sarah E; Marky, Luis A

2016-05-01

The overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. To investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH(+)) in the helix-coil transition of DNA molecules. We use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. We determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C(+)GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition. Copyright © 2015. Published by Elsevier B.V.

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

PubMed Central

Stone, G. E.; Miller, O. L.; Prescott, D. M.

1965-01-01

The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Delineating the cell death mechanisms associated with skin electroporation.

PubMed

Schultheis, Katherine; Smith, Trevor R F; Kiosses, William B; Kraynyak, Kimberly A; Wong, Amelia; Oh, Janet; Broderick, Kate Elizabeth

2018-06-28

The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of pDNA into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses. Here, we have investigated the possible mechanisms associated with this adjuvant effect, primarily focusing on the cell death pathways associated with the skin EP procedure independent of pDNA delivery. Using the minimally invasive CELLECTRA®-3P intradermal electroporation device that penetrates the epidermal and dermal layers of the skin, we have investigated apoptotic and necrotic cell death in relation to the vicinity of the electrode needles and electric field generated. Employing the well-established TUNEL assay, we detected apoptosis beginning as early as one hour after EP and peaking at the 4 hour time point. The majority of the apoptotic events were detected in the epidermal region directly adjacent to the electrode needle. Using a novel propidium iodide in vivo necrotic cell death assay, we detected necrotic events concentrated in the epidermal region adjacent to the electrode. Furthermore, we detected up-regulation of calreticulin expression on skin cells after EP, thus labeling these cells for uptake by dendritic cells and macrophages. These results allow us to delineate the cell death mechanisms occurring in the skin following intradermal EP independently of pDNA delivery. We believe these events contribute to the adjuvant effect observed following electroporation at the skin treatment site.

Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

NASA Astrophysics Data System (ADS)

Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

2007-12-01

Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10 ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability of 60-70% of the positive control.

Characterization of rabies pDNA nanoparticulate vaccine in poloxamer 407 gel.

PubMed

Bansal, Amit; Wu, Xianfu; Olson, Victoria; D'Souza, Martin J

2018-07-10

Plasmid DNA (pDNA) vaccines have the potential for protection against a wide range of diseases including rabies but are rapid in degradation and poor in uptake by antigen-presenting cells. To overcome the limitations, we fabricated a pDNA nanoparticulate vaccine. The negatively charged pDNA was adsorbed onto the surface of cationic PLGA (poly (d, l-lactide-co-glycolide))-chitosan nanoparticles and were used as a delivery vehicle. To create a hydrogel for sustainable vaccine release, we dispersed the pDNA nanoparticles in poloxamer 407 gel which is liquid at 4 °C and turns into soft gels at 37 °C, providing ease of administration and preventing burst release of pDNA. Complete immobilization of pDNA to cationic nanoparticles was achieved at a pDNA to nanoparticles ratio (P/N) of 1/50. Cellular uptake of nanoparticles was both time and concentration dependent and followed a saturation kinetics with V max of 11.389 µg/mL h and K m of 139.48 µg/mL. The in vitro release studies showed the nanoparticulate vaccine has a sustained release for up to 24 days. In summary, pDNA PLGA-chitosan nanoparticles were non-cytotoxic, their buffering capacity and cell uptake were enhanced, and sustained the release of pDNA. We expect our pDNA vaccine's potency will be greatly improved in the animal studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.

PubMed

Tu, Thomas; Budzinska, Magdalena A; Vondran, Florian W R; Shackel, Nicholas A; Urban, Stephan

2018-02-07

Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. Importance Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation. Copyright © 2018 American Society for Microbiology.

Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission of Babesia parasite.

PubMed

Boldbaatar, Damdinsuren; Battsetseg, Badgar; Matsuo, Tomohide; Hatta, Takeshi; Umemiya-Shirafuji, Rika; Xuan, Xuenan; Fujisaki, Kozo

2008-08-01

A cDNA encoding the vitellogenin receptor of the ixodid tick, Haemaphysalis longicornis Neumann (HlVgR) was cloned and characterized. The full-length cDNA is 5631 bp, including an intact ORF encoding an expected protein with 1782 amino acids. The deduced amino acid sequence of the HlVgR cDNA revealed two ligand-binding domains with four class A cysteine-rich repeats in the first domain and eight in the second domain similar to those of insect VgRs. The immunoblot analysis detected approximately 197 kDa protein in both tick ovary and egg. The developmental expression profile demonstrated that HlVgR mRNA exists throughout the ovarian development, and the transcriptional level is especially high in the previtellogenic period. Immuno electron microscopy analysis demonstrated that the localization of HlVgR is detected on the external surface of oocyte plasma membrane. RNAi showed that eggs of HlVgR dsRNA-injected adult ticks had not developed into fully mature oocytes and laid abnormal eggs. The Babesia parasite DNA was not detected in the eggs of HlVgR dsRNA-injected tick that fed on Babesia gibsoni infected dog, whereas it was detected in the eggs of PBS-injected ticks and noninjected ticks. Expression of HlVgR was increased by the vitellogenic hormone 20-hydroxyecdysone. These results indicate that HlVgR, which is produced by the developing oocytes, is essential for Vg uptake, egg development in the H. longicornis tick, and transovarial transmission of Babesia parasites.

Positive view and increased likely uptake of follow-up testing with analysis of cell-free fetal DNA as alternative to invasive testing among Danish pregnant women.

PubMed

Miltoft, Caroline B; Rode, Line; Tabor, Ann

2018-05-01

The aim of this study was to investigate the attitude (view, likely uptake and preferred strategy) towards cell-free fetal DNA (cfDNA) testing among pregnant women before a first-trimester risk assessment for trisomy 21 (unselected women) and after obtaining a high risk. Unselected and high-risk women attending first-trimester screening (Rigshospitalet, Copenhagen University Hospital) were invited to fill out the questionnaire Antenatal testing for Down syndrome as an online survey. The survey included 203 unselected and 50 high-risk women (response rates of 74.8% and 84.7%, respectively). Nearly all considered cfDNA testing a positive development in antenatal care, and 97.2% would like it to be offered. Offering cfDNA testing as an alternative to invasive testing would increase the uptake of follow-up testing compared with invasive testing alone (98.8% vs. 90.7%, p < 0.001). Women who would only accept follow up by cfDNA testing were more likely to continue an affected pregnancy (30.0% vs. 3.6%, p < 0.001) or have doubts about termination (50.0% vs. 32.1%, p < 0.001). Offering cfDNA testing would likely increase the uptake of follow-up testing without a corresponding rise in the termination rate of affected fetuses as some women test for information only. However, both unselected and high-risk women had overwhelmingly positive views underlining attention to avoid routinization. © 2018 Nordic Federation of Societies of Obstetrics and Gynecology.

G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

NASA Astrophysics Data System (ADS)

Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

2016-10-01

The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

PubMed Central

Lauter, F R; Ninnemann, O; Bucher, M; Riesmeier, J W; Frommer, W B

1996-01-01

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8755617

The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, K.; Borovilos, M.; Zhou, M

2009-12-25

Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representingmore » a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.« less

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

PubMed Central

Usdin, K; Furano, A V

1988-01-01

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed. Images PMID:2837766

The extent of the uptake of plasmid into the skin determines the immune responses induced by a DNA vaccine applied topically onto the skin

PubMed Central

Yu, Zhen; Chung, Woon-Gye; Sloat, Brian R.; Löhr, Christiane V.; Weiss, Richard; Rodriguez, B. Leticia; Li, Xinran; Cui, Zhengrong

2011-01-01

Objectives Non-invasive immunization by applying plasmid DNA topically onto the skin is an attractive immunization approach. However, the immune responses induced are generally weak. Previously, we showed that the antibody responses induced by topical DNA vaccine were significantly enhanced when hair follicles in the application area were induced into anagen (growth) stage by hair plucking. In the present study, we further investigated the mechanism of immune enhancement. Methods Three different methods, hair plucking or treatment with retinoic acid (RA) or O- tetradecanoylphorbol-13-acetate (TPA), were used to induce hair follicles into anagen stage before mice were dosed with a β-galactosidase-encoding plasmid, and the specific antibody responses induced were evaluated. Key findings The hair plucking method was more effective at enhancing the resultant antibody responses. Treatment with RA or TPA caused more damages to the skin and induced more severe local inflammations than hair plucking. However, hair plucking was most effective at enhancing the uptake or retention of the DNA in the application area. Conclusions The uptake of plasmid DNA in the application area correlated with the antibody responses induced by a topically applied DNA. PMID:21235583

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

PubMed Central

Lopata, M A; Cleveland, D W; Sollner-Webb, B

1984-01-01

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA. Images PMID:6589587

Targeted transplantation of mitochondria to hepatocytes

PubMed Central

Gupta, Nidhi; Wu, Catherine H; Wu, George Y

2016-01-01

Background Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. Materials and methods An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. Results Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. Conclusion Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis. PMID:27942238

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

NASA Astrophysics Data System (ADS)

Zhang, Guiyu; Zhang, Zhiyong; Yang, Junen

2017-08-01

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency.

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

PubMed

Zheng, Hao; Tang, Cui; Yin, Chunhua

2015-06-01

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

NASA Astrophysics Data System (ADS)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-03-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

PubMed

Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

2017-02-01

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr + ][Ntf 2 - ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

Labeling TiO2 nanoparticles with dyes for optical fluorescence microscopy and determination of TiO2-DNA nanoconjugate stability.

PubMed

Thurn, Kenneth T; Paunesku, Tatjana; Wu, Aiguo; Brown, Eric M B; Lai, Barry; Vogt, Stefan; Maser, Jörg; Aslam, Mohammed; Dravid, Vinayak; Bergan, Raymond; Woloschak, Gayle E

2009-06-01

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO2) nanoparticles. These nanoparticles, when electronically linked to single-stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO2-DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO2 nanoparticles and TiO2-DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO2 nanoparticle uptake in a large population of living cells (>10(4) cells). X-ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO2-DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO2-DNA nanoconjugates in malignant cells.

Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery.

PubMed

Bahadoran, Azadeh; Moeini, Hassan; Bejo, Mohd Hair; Hussein, Mohd Zobir; Omar, Abdul Rahman

In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-).  In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine.  The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum. PMID:20055984

The disaccharide moiety of bleomycin facilitates uptake by cancer cells.

PubMed

Schroeder, Benjamin R; Ghare, M Imran; Bhattacharya, Chandrabali; Paul, Rakesh; Yu, Zhiqiang; Zaleski, Paul A; Bozeman, Trevor C; Rishel, Michael J; Hecht, Sidney M

2014-10-01

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

Cationic nanoemulsions as potential carriers for intracellular delivery

PubMed Central

Khachane, P.V.; Jain, A.S.; Dhawan, V.V.; Joshi, G.V.; Date, A.A.; Mulherkar, R.; Nagarsenker, M.S.

2014-01-01

Successful cytosolic delivery enables opportunities for improved treatment of various genetic disorders, infectious diseases and cancer. Cationic nanoemulsions were designed using alternative excipients and evaluated for particle size, charge, effect of sterilization on its stability, DNA condensation potential and cellular uptake efficiency. Various concentrations of non-ionic and ionic stabilizers were evaluated to design formula for colloidally stable cationic nanoemulsion. The nanoemulsion comprised of 5% Capmul MCM, 0.5% didodecyldimethylammonium bromide (DDAB), 1% phospholipid, 1% Poloxamer 188 and 2.25% glycerol and possessed particle size of 81.6 ± 3.56 nm and 137.1 ± 1.57 nm before and after steam sterilization, respectively. DNA condensation studies were carried out at various nanoemulsion: DNA ratios ranging from 1:1 to 10:1. Cell uptake studies were conducted on human embryonic kidney (HEK) cell lines which are widely reported for transfection studies. The nanoemulsions showed excellent cellular uptake as evaluated by fluorescence microscopy and flow cytometry. Overall, a colloidally stable cationic nanoemulsion with good DNA condensation ability was successfully fabricated for efficient cytosolic delivery and potential for in vivo effectiveness. PMID:25972740

Design and Application of Multifunctional DNA Nanocarriers for Therapeutic Delivery

PubMed Central

Charoenphol, Phapanin; Bermudez, Harry

2013-01-01

The unique programmability of nucleic acids offers versatility and flexibility in the creation of self-assembled DNA nanostructures. To date, many three-dimensional DNA architectures have been precisely formed of varying sizes and shapes. Their biocompatibility, biodegradability, and high intrinsic stability in physiological environments emphasize their emerging use as carriers for drug and gene delivery. Furthermore, DNA nanocarriers have been shown to enter cells efficiently and without the aid of transfection reagents. A key strength of DNA nanocarriers over other delivery systems is their modularity and their ability to control the spatial distribution of cargoes and ligands. Optimizing DNA nanocarrier properties to dictate their localization, uptake, and intracellular trafficking is also possible. In this review, we present design considerations for DNA nanocarriers and examples of their use in the context of therapeutic delivery applications. The assembly of DNA nanocarriers and approaches for loading and releasing cargo are described. The stability and safety of DNA nanocarriers is also discussed, with particular attention to the in vivo physiological environment. Mechanisms of cellular uptake and intracellular trafficking are examined, and we conclude with strategies to enhance the delivery efficiency of DNA nanocarriers. PMID:23896566

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.

PubMed

Kodama, Yukinobu; Ohkubo, Chikako; Kurosaki, Tomoaki; Egashira, Kanoko; Sato, Kayoko; Fumoto, Shintaro; Nishida, Koyo; Higuchi, Norihide; Kitahara, Takashi; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

Seventeen Sxy-Dependent Cyclic AMP Receptor Protein Site-Regulated Genes Are Needed for Natural Transformation in Haemophilus influenzae

PubMed Central

Mell, Joshua C.; Redfield, Rosemary J.

2012-01-01

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized. PMID:22821979

Seventeen Sxy-dependent cyclic AMP receptor protein site-regulated genes are needed for natural transformation in Haemophilus influenzae.

PubMed

Sinha, Sunita; Mell, Joshua C; Redfield, Rosemary J

2012-10-01

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

PubMed Central

Mezquita, C; Teng, C S

1977-01-01

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 PMID:560187

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

PubMed

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W; Shah, Manali; Glenn, Kevin C; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.

Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

PubMed

Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

2015-08-01

DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice.

PubMed

Khazanov, Elena; Simberg, Dmitri; Barenholz, Yechezkel

2006-08-01

Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflammatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy. 2006 John Wiley & Sons, Ltd.

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes

PubMed Central

Aizawa, Shu; Contu, Viorica Raluca; Fujiwara, Yuuki; Hase, Katsunori; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji

2017-01-01

ABSTRACT Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy. PMID:27846365

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

PubMed

Aizawa, Shu; Contu, Viorica Raluca; Fujiwara, Yuuki; Hase, Katsunori; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2017-01-02

Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

PubMed Central

Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

2015-01-01

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

PubMed Central

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

NASA Astrophysics Data System (ADS)

Miller, Cynthia K.; Temin, Howard M.

1983-05-01

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Simulation model of converging-diverging (CD) nozzle to improve particle delivery system of deoxyribonucleic acid (DNA)

NASA Astrophysics Data System (ADS)

Sumarsono, Danardono A.; Ibrahim, Fera; Santoso, Satria P.; Sari, Gema P.

2018-02-01

Gene gun is a mechanical device which has been used to deliver DNA vaccine into the cells and tissues by increasing the uptake of DNA plasmid so it can generate a high immune response with less amount of DNA. Nozzle is an important part of the gene gun which used to accelerate DNA in particle form with a gas flow to reach adequate momentum to enter the epidermis of human skin and elicit immune response. We developed new designs of nozzle for gene gun to make DNA uptake more efficient in vaccination. We used Computational Fluid Dynamics (CFD) by Autodesk® Simulation 2015 to simulate static fluid pressure and velocity contour of supersonic wave and parametric distance to predict the accuracy of the new nozzle. The result showed that the nozzle could create a shockwave at the distance parametric to the object from 4 to 5 cm using fluid pressure varied between 0.8-1.2 MPa. This is indication a possibility that the DNA particle could penetrate under the mammalian skin. For the future research step, this new nozzle model could be considered for development the main component of the DNA delivery system in vaccination in vivo

Examining multi-component DNA-templated nanostructures as imaging agents

NASA Astrophysics Data System (ADS)

Jaganathan, Hamsa

2011-12-01

Magnetic resonance imaging (MRI) is the leading non-invasive tool for disease imaging and diagnosis. Although MRI exhibits high spatial resolution for anatomical features, the contrast resolution is low. Imaging agents serve as an aid to distinguish different types of tissues within images. Gadolinium chelates, which are considered first generation designs, can be toxic to health, while ultra-small, superparamagnetic nanoparticles (NPs) have low tissue-targeting efficiency and rapid bio-distribution, resulting to an inadequate detection of the MRI signal and enhancement of image contrast. In order to improve the utility of MRI agents, the challenge in composition and structure needs to be addressed. One-dimensional (1D), superparamagnetic nanostructures have been reported to enhance magnetic and in vivo properties and therefore has a potential to improve contrast enhancement in MRI images. In this dissertation, the structure of 1D, multi-component NP chains, scaffolded on DNA, were pre-clinically examined as potential MRI agents. First, research was focused on characterizing and understanding the mechanism of proton relaxation for DNA-templated NP chains using nuclear magnetic resonance (NMR) spectrometry. Proton relaxation and transverse relaxivity were higher in multi-component NP chains compared to disperse NPs, indicating the arrangement of NPs on a 1D structure improved proton relaxation sensitivity. Second, in vitro evaluation for potential issues in toxicity and contrast efficiency in tissue environments using a 3 Tesla clinical MRI scanner was performed. Cell uptake of DNA-templated NP chains was enhanced after encapsulating the nanostructure with layers of polyelectrolytes and targeting ligands. Compared to dispersed NPs, DNA-templated NP chains improved MRI contrast in both the epithelial basement membrane and colon cancer tumors scaffolds. The last part of the project was focused on developing a novel MRI agent that detects changes in DNA methylation levels. The findings from this dissertation suggest that the structural arrangement of NPs on DNA significantly influenced their function and utility as MRI agents.

Adsorption of plasmid DNA on anion exchange chromatography media.

PubMed

Tarmann, Christina; Jungbauer, Alois

2008-08-01

Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 A 30 microm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of self-assembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea.

PubMed

Leonhardt, Tereza; Sácký, Jan; Kotrba, Pavel

2018-04-01

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1 H208A or RaZIP1 H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Re-induction of cell differentiation and (131)I uptake in dedifferentiated FTC-133 cell line by TSHR gene transfection.

PubMed

Feng, Fang; Wang, Hui; Hou, Shasha; Fu, Hongliang

2012-11-01

Radioiodine therapy is commonly used to treat differentiated thyroid cancer (DTC), but a major challenge is dedifferentiation of DTC with the loss of radioiodine uptake. TSHR is a key molecule regulating thyrocyte proliferation and function. This study aimed to test the therapeutic potential of TSHR in dedifferentiated DTC by gene transfection in order to restore cell differentiation and radioiodine uptake. Dedifferentiated FTC-133 (dFTC-133) cells were obtained by monoclonal culture of FTC-133 cell line after (131)I radiation. Recombinant plasmid pcDNA3.1-hTSHR was transfected into dFTC-133 cells by using Lipofectamine 2000 reagent. Immunofluorescence analysis was carried out to confirm TSHR expression and its location. Radioiodine uptake assay was thereafter investigated. mRNAs and proteins of TSHR and other thyroid differentiated markers were detected by real-time PCR and western blot respectively. Among the thyroid specific genes in dFTC-133 cells with stable low radioiodine uptake, TSHR was down-regulated most significantly compared with FTC-133. Then, after TSHR gene transfection, augmented expression of TSHR was observed in dFTC-133 cell surface and cytoplasm by immunofluorescence analysis. It was found that (125)I uptake was 2.9 times higher (t=28.63, P<.01) in cells with TSHR transfection than control. The mRNAs of TSHR, NIS, TPO and Tg were also significantly increased by 1.7 times (t=13.8, P<.05), 4 times (t=28.52, P<.05), 1.5 times (t=14.43, P<.05) and 2.2 times (t=19.83, P<.05) respectively compared with control group. Decreased TSHR expression correlated with FTC-133 ongoing dedifferentiation. TSHR transfection contributed to the re-differentiation of dedifferentiated thyroid follicular carcinoma cells. Copyright © 2012 Elsevier Inc. All rights reserved.

A physiological and genetic approach to the improvement of tomato (Lycopersicon esculentum Mill. ) fruit soluble solids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Damon, S.E.

Physiological processes and the genetic basis determining soluble solids content (SSC) of processing tomato fruit were addressed. Analysis of ({sup 3}H)-(fructosyl)-sucrose translocation in tomato indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast. Apoplastic sucrose, glucose and fructose concentrations were estimated as 1 to 7, 12 to 49 and 8 to 63 millimolar, respectively in tomato fruit pericarp. Short-term uptake of ({sup 14}C)sucrose, -glucose and -fructose in tomato pericarp discs showes first order kinetics over the physiologically relevant concentration range. The uptake of ({sup 14}C)-(glycosyl)-1{prime}fluorosucrose was identical to the rate of ({sup 14}C) sucrosemore » uptake suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Sugar uptake across the plasmamembrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast. Fourteen genomic DNA probes and ten restriction endonucleases were used to identify restriction fragment length polymorphisms (RFLPs) useful in the linkage analysis of quantitative trait loci controlling the expression of SSC in a segregating F{sub 2} population from a cross between L. esculentum (UC204B) and L. cheesmanii f. minor, a wild species with high fruit soluble solids. RFLPs were detected between the DNAs of the two tomato species with all 14 probes.« less

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

PubMed

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

PubMed Central

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter.

PubMed

Bes, M T; Hernández, J A; Peleato, M L; Fillat, M F

2001-01-15

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

PubMed Central

Peng, Rong; Maklokova, Vilena I.; Chandrashekhar, Jayadevi H.; Lan, Que

2011-01-01

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes. PMID:21437205

Suppression of phytohemagglutinin-induction of thymidine uptake in guinea pig lymphocytes by methylglyoxal bis(guanylhydrazone) treatment.

PubMed

Otani, S; Matsui, I; Morisawa, S

1977-10-18

Treatment with methylglyoxal bis(guanylhydrazone), a specific inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), suppressed the phytohemagglutinin-induction of [3H]thymidine uptake by guinea pig lymphocytes. The kinetics of [3H]thymidine uptake revealed that the Km value for thymidine was not changed, but the V value was markedly lowered by the methylglyoxal bis(guanylhydrazone) treatment. The induction of ATP: thymidine 5'-phosphotransferase (EC 2.7.1.75) (thymidine kinase) activity by phytohemagglutinin was suppressed to about the same extent as the induction of thymidine uptake. These suppressions were dependent on the methylglyoxal bis(guanylhydrazone) doses and on duration of the methylglyoxal bis(guanylhydrazone) treatment. Analysis of [3H]thymidine labelled compounds of the acid-soluble fraction showed that conversion of thymidine to thymidine 5'-triphosphate was inhibited by the methylglyoxal bis(guanylhydrazone) treatment. DNA polymerase activity was less inhibited by the methylglyoxal bis(guanylhydrazone) treatment in comparison with the methylglyoxal bis(guanylhydrazone) inhibition of thymidine uptake by whole cells. These results strongly suggested that blocking of polyamine accumulation by the methylglyoxal bis(guanylhydrazone) treatment influenced phytohemagglutinin induction of thymidine phosphorylation, resulting in a decrease of thymidine incorporation into DNA.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras.

PubMed

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-08-09

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras

PubMed Central

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-01-01

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients. PMID:27437771

Zinc as a second messenger of mitogenic induction. Effects on diadenosine tetraphosphate (Ap4A) and DNA synthesis.

PubMed

Grummt, F; Weinmann-Dorsch, C; Schneider-Schaulies, J; Lux, A

1986-03-01

DNA synthesis and adenosine(5')tetraphosphate(5')adenosine (Ap4A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micromolar amounts of ZnCl2. ZnCl2 in micromolar concentrations also inhibits Ap4A hydrolase and stimulates amino acid-dependent Ap4A synthesis, suggesting that Zn2+ is modulating intracellular Ap4A pools. Serum addition to G1-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap4A as a possible 'third messenger' and trigger of DNA synthesis.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples

PubMed Central

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W.; Shah, Manali; Glenn, Kevin C.; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. PMID:27314015

Enhanced cell uptake via non-covalent decollation of a single-walled carbon nanotube-DNA hybrid with polyethylene glycol-grafted poly(l-lysine) labeled with an Alexa-dye and its efficient uptake in a cancer cell

NASA Astrophysics Data System (ADS)

Fujigaya, Tsuyohiko; Yamamoto, Yuki; Kano, Arihiro; Maruyama, Atsushi; Nakashima, Naotoshi

2011-10-01

The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found.The use of single-walled carbon nanotubes (SWNTs) for biomedical applications is a promising approach due to their unique outer optical stimuli response properties, such as a photothermal response triggered by near-IR laser irradiation. The challenging task in order to realize such applications is to render the SWNTs biocompatible. For this purpose, the stable and homogeneous functionalization of the SWNTs with a molecule carrying a biocompatible group is very important. Here, we describe the design and synthesis of a polyanionic SWNT/DNA hybrid combined with a cationic poly(l-lysine) grafted by polyethylene glycol (PLL-g-PEG) to provide a supramolecular SWNT assembly. A titration experiment revealed that the assembly undergoes an approximately 1 : 1 reaction of the SWNT/DNA with PLL-g-PEG. We also found that SWNT/DNA is coated with PLL-g-PEG very homogeneously that avoids the non-specific binding of proteins on the SWNT surface. The experiment using the obtained supramolecular hybrid was carried out in vitro and a dramatic enhancement in the cell uptake efficiency compared to that of the SWNT/DNA hybrid without PLL-g-PEG was found. Electronic supplementary information (ESI) available: Additional absorption spectra, DLS plots and PL spectra. See DOI: 10.1039/c1nr10635j

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine.

PubMed

Welter, Harald; Claus, Rolf

2008-06-01

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.

A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

PubMed

Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

2017-08-01

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

PubMed

Barnard, Anna; Posocco, Paola; Pricl, Sabrina; Calderon, Marcelo; Haag, Rainer; Hwang, Mark E; Shum, Victor W T; Pack, Daniel W; Smith, David K

2011-12-21

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery. © 2011 American Chemical Society

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

PubMed

Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

2017-11-01

Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of genes involved in the uptake of inorganic carbon in the gill of a deep-sea vesicomyid clam harboring intracellular thioautotrophic bacteria.

PubMed

Hongo, Yuki; Ikuta, Tetsuro; Takaki, Yoshihiro; Shimamura, Shigeru; Shigenobu, Shuji; Maruyama, Tadashi; Yoshida, Takao

2016-07-10

Deep-sea vesicomyid clams, including the genus Phreagena (formerly Calyptogena), harbor thioautotrophic bacterial symbionts in the host symbiosome, which consists of cytoplasmic vacuoles in gill epithelial cells called bacteriocytes. The symbiont requires inorganic carbon (Ci), such as CO2, HCO3(-), and CO3(2-), to synthesize organic compounds, which are utilized by the host clam. The dominant Ci in seawater is HCO3(-), which is impermeable to cell membranes. Within the bacteriocyte, cytoplasmic carbonic anhydrase (CA) from the host, which catalyzes the inter-conversion between CO2 and HCO3(-), has been shown to be abundant and is thought to supply intracellular CO2 to symbionts in the symbiosome. However, the mechanism of Ci uptake by the host gill from seawater is poorly understood. To elucidate the influx pathway of Ci into the bacteriocyte, we isolated the genes related to Ci uptake via the pyrosequencing of cDNA from the gill of Phreagena okutanii, and investigated their expression patterns. Using phylogenetic and amino acid sequence analyses, three solute carrier family 4 (SLC4) bicarbonate transporters (slc4co1, slc4co2, and slc4co4) and two membrane-associated CAs (mcaco1 and mcaco2) were identified as candidate genes for Ci uptake. In an in situ hybridization analysis of gill sections, the expression of mcaco1 and mcaco2 was detected in the bacteriocytes and asymbiotic non-ciliated cells, respectively, and the expression of slc4co1 and slc4co2 was detected in the asymbiotic cells, including the intermediate cells of the inner area and the non-ciliated cells of the external area. Although subcellular localizations of the products of these genes have not been fully elucidated, they may play an important role in the uptake of Ci into the bacteriocytes. These findings will improve our understanding of the Ci transport system in the symbiotic relationships of chemosynthetic bivalves. Copyright © 2016 Elsevier B.V. All rights reserved.

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method.

PubMed

Lehtinen, Julia; Hyvönen, Zanna; Subrizi, Astrid; Bunjes, Heike; Urtti, Arto

2008-10-21

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Functional analysis of the ComK protein of Bacillus coagulans.

PubMed

Kovács, Ákos T; Eckhardt, Tom H; van Hartskamp, Mariska; van Kranenburg, Richard; Kuipers, Oscar P

2013-01-01

The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis- and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNA-uptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B.subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis.

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

PubMed Central

Ledinko, Nada; Fong, Caroline K. Y.

1969-01-01

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of 3H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. 3H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much 3H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase. PMID:5806981

Electrostimulation of rat callus cells and human lymphocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aro, H.; Eerola, E.; Aho, A.J.

1984-01-01

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took upmore » more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.« less

Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblasts.

PubMed

Vassbotn, F S; Langeland, N; Holmsen, H

1990-09-01

Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.

Vasopressin stimulates DNA synthesis and ion transport in quiescent epithelial cells.

PubMed

Reznik, V M; Shapiro, R J; Mendoza, S A

1985-09-01

The mitogenic effect of vasopressin was studied in subconfluent quiescent renal epithelial cells (MDCK). Vasopressin stimulated DNA synthesis in the presence of low concentrations of serum. Vasopressin increased the entry of Na into the cells and increased ouabain-sensitive 86Rb uptake, a measure of Na-K pump activity. Because the activity of the Na-K pump in MDCK cells is steeply dependent on intracellular Na, it is likely that stimulation of the Na-K pump by vasopressin was mediated by the increase in Na entry into the cells. Thus both serum and vasopressin stimulate Na uptake and Na-K pump activity in quiescent MDCK cells with a subsequent increase in DNA synthesis. It is concluded that growth regulation in epithelial cells may be mediated in part by changes in monovalent ion transport.

Smart Carbon Nanotubes with Laser-Controlled Behavior in Gene Delivery and Therapy through a Non-Digestive Trafficking Pathway.

PubMed

Kong, Fenfen; Liu, Fei; Li, Wei; Guo, Xiaomeng; Wang, Zuhua; Zhang, Hanbo; Li, Qingpo; Luo, Lihua; Du, Yongzhong; Jin, Yi; You, Jian

2016-12-01

Near-infrared (NIR) laser-controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a "photothermal transfection" agent is obtained by wrapping poly(ethylenimine)-cholesterol derivatives (PEI-Chol) around single-walled carbon nanotubes (SWNTs). The PEI-Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae-mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser-mediated photothermal transfection of PCS 10K /plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor-growth inhibition in vivo than naked pTP53, PEI 25K /pTP53, and PCS 10K /pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae-mediated cellular uptake of the complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human β‐defensin 3 increases the TLR9‐dependent response to bacterial DNA

PubMed Central

McGlasson, Sarah L.; Semple, Fiona; MacPherson, Heather; Gray, Mohini; Davidson, Donald J.

2017-01-01

Human β‐defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self‐DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt‐3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self‐DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9‐dependent response. In contrast to hBD3, lipofection of self‐DNA enhances inflammatory signaling, but the response is predominantly TLR9‐independent. Together, these data show that hBD3 has a role in the innate immune‐mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy‐number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA‐based vaccines. PMID:28102569

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

PubMed

Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

2016-03-15

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, F.A.; Gray, J.W.

1983-10-18

A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

PubMed

Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

2014-01-15

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Human β-defensin 3 increases the TLR9-dependent response to bacterial DNA.

PubMed

McGlasson, Sarah L; Semple, Fiona; MacPherson, Heather; Gray, Mohini; Davidson, Donald J; Dorin, Julia R

2017-04-01

Human β-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self-DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt-3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self-DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9-dependent response. In contrast to hBD3, lipofection of self-DNA enhances inflammatory signaling, but the response is predominantly TLR9-independent. Together, these data show that hBD3 has a role in the innate immune-mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy-number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA-based vaccines. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1986-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

Activation of different split functionalities upon re-association of RNA-DNA hybrids

PubMed Central

Afonin, Kirill A.; Viard, Mathias; Martins, Angelica N.; Lockett, Stephen J.; Maciag, Anna E.; Freed, Eric O.; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A.

2013-01-01

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications. PMID:23542902

The Rate and Clinical Significance of Incidental Thyroid Uptake as Detected by Gallium-68 DOTATATE Positron Emission Tomography/Computed Tomography

PubMed Central

Nockel, Pavel; Millo, Corina; Keutgen, Xavier; Klubo-Gwiezdzinska, Joanna; Shell, Jasmine; Patel, Dhaval; Nilubol, Naris; Herscovitch, Peter; Sadowski, Samira M.

2016-01-01

Background: Gallium-68 (Ga-68) DOTATATE is a radiolabeled peptide–imaging modality that targets the somatostatin receptor (SSTR), especially subtype 2 (SSTR2). Benign and malignant thyroid tumors have been observed to express SSTR. The aim of this study was to evaluate the frequency and clinical significance of incidental atypical thyroid uptake as detected by Ga-68 DOTATATE positron emission tomography/computed tomography (PET/CT). Methods: A retrospective analysis was conducted of a prospective study in which 237 patients underwent Ga-68 DOTATATE PET/CT as part of a work-up for metastatic and unknown primary neuroendocrine tumors. The types of uptake in the thyroid gland (focal/diffuse) and maximum standardized uptake value (SUVmax) levels were evaluated and compared with the background uptake in the liver and salivary glands. Results: Of 237 patients, 26 (11%) had atypical thyroid uptake as detected by Ga-68 DOTATATE PET/CT. There were no significant clinical or biochemical variables associated with atypical thyroid uptake. Fourteen (54%) patients had positive focal uptake, and 12 (46%) patients had diffuse uptake. Of the 14 patients with atypical focal uptake, 10 (71%) had thyroid nodules on the corresponding side, as detected by anatomic imaging. Three of 10 patients (21%) were found to have papillary thyroid cancer, and seven (70%) had adenomatoid nodules. Of the 12 patients with diffuse increased uptake, six (50%) had a history of hypothyroidism, five (42%) had chronic lymphocytic thyroiditis, and one (8%) had nontoxic multinodular goiter. Conclusions: Patients with an incidental focal abnormal thyroid uptake on Ga-68 DOTATATE PET/CT scan should have further clinical evaluation to exclude a diagnosis of thyroid cancer. PMID:27094616

Follow-up bone scan in breast cancer patients: what is the appropriate interpretation of purely rib uptake?

PubMed

Seo, Minjung; Ko, Byung Kyun; Tae, Soon Young; Koh, Su-Jin; Noh, Young Ju; Choi, Hye-Jeong; Bae, Kyungkyg; Bang, Minseo; Jun, Sungmin; Park, Seol Hoon

2016-12-01

Although rib uptake is frequently detected in follow-up bone scans of breast cancer patients, few studies have assessed its clinical significance. Among 1208 breast cancer patients who underwent a bone scan between 2011 and 2014, 157 patients presented with newly detected rib uptake at follow-up. Patients who had underlying bone metastases (n=8) or had simultaneous new uptake in sites other than the rib (n=13) were excluded. The patients enrolled finally were those who had purely rib uptakes. The location, intensity, and final diagnosis of the uptake were evaluated by nuclear medicine physicians. A total of 275 new instances of rib uptake were detected in follow-up bone scans of 136 patients. These were more frequently located on the ipsilateral side of the breast cancer (61.1%) and the anterior arc (65.1%), and they presented as moderate to intense (93.1%) uptakes. Among these, 265 lesions in 130 patients turned out to be benign fractures (96.4%), whereas only 10 lesions in six patients were metastases. The proportion of metastases was significantly higher if the uptake was linear or if the patient had recurrence. It was marginally higher if the uptake was located in the posterior arc. The proportion of metastases within the radiation field was significantly lower in patients with a history of irradiation. Newly detected purely rib uptake on a follow-up bone scan in patients who have been treated for breast cancer is mostly because of fractures and rarely signals metastasis. However, if the patient has disease recurrence, metastasis should strongly be suspected, particularly when uptake is linear or located in the posterior arc.

Genotyping analysis and ¹⁸FDG uptake in breast cancer patients: a preliminary research.

PubMed

Bravatà, Valentina; Stefano, Alessandro; Cammarata, Francesco P; Minafra, Luigi; Russo, Giorgio; Nicolosi, Stefania; Pulizzi, Sabina; Gelfi, Cecilia; Gilardi, Maria C; Messa, Cristina

2013-04-30

Diagnostic imaging plays a relevant role in the care of patients with breast cancer (BC). Positron Emission Tomography (PET) with 18F-fluoro-2-deoxy-D-glucose (FDG) has been widely proven to be a clinical tool suitable for BC detection and staging in which the glucose analog supplies metabolic information about the tumor. A limited number of studies, sometimes controversial, describe possible associations between FDG uptake and single nucleotide polymorphisms (SNPs). For this reason this field has to be explored and clarified. We investigated the association of SNPs in GLUT1, HIF-1a, EPAS1, APEX1, VEGFA and MTHFR genes with the FDG uptake in BC. In 26 caucasian individuals with primary BC, whole-body PET-CT scans were obtained and quantitative analysis was performed by calculating the maximum Standardized Uptake Value normalized to body-weight (SUVmax) and the mean SUV normalized to body-weight corrected for partial volume effect (SUVpvc). Human Gene Mutation Database and dbSNP Short Genetic Variations database were used to analyze gene regions containing the selected SNPs. Patient genotypes were obtained using Sanger DNA sequencing analysis performed by Capillary Electrophoresis. BC patients were genotyped for the following nine SNPs: GLUT1: rs841853 and rs710218; HIF-1a: rs11549465 and rs11549467; EPAS1: rs137853037 and rs137853036; APEX1: rs1130409; VEGFA: rs3025039 and MTHFR: rs1801133. In this work correlations between the nine potentially useful polymorphisms selected and previously suggested with tracer uptake (using both SUVmax and SUVpvc) were not found. The possible functional influence of specific SNPs on FDG uptake needs further studies in human cancer. In summary, this is the first pilot study, to our knowledge, which investigates the association between a large panel of SNPs and FDG uptake specifically in BC patients. This work represents a multidisciplinary and translational medicine approach to study BC where, the possible correlation between SNPs and tracer uptake, may be considered to improve personalized cancer treatment and care.

Nucleases Encoded by Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

The species Campylobacter jejuni displays huge genetic diversity, and is naturally competent for DNA uptake. Nevertheless, not every strain is able to acquire foreign DNA since nonnaturally transformable strains do exist. Previously we showed that many nonnaturally transformable C. jejuni strains ex...

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT.

PubMed

Sánchez-Villalba, Esther; Arias, María Elena; Zambrano, Fabiola; Loren, Pía; Felmer, Ricardo

2018-02-01

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

OBSERVATIONS ON THE UPTAKE OF TRITIATED THYMIDINE IN THE PRONUCLEI OF FERTILIZED SAND DOLLAR EMBRYOS.

PubMed

Simmel, E B; Karnofsky, D A

1961-05-01

Following fertilization of the egg of the sand dollar Echinarachnius parma, tritiated thymidine (H(3)TDR) was taken up independently by the male and female pronuclei beginning within about 15 to 20 minutes, and the labeled pronuclei fused at about 30 to 40 minutes. At cleavage 90 minutes later the labeled nuclear material was distributed to both daughter cells. Unfertilized eggs and sperm exposed to H(3)TDR did not show nuclear localization of thymidine. DNA replication, thus, is initiated in the haploid pronuclei shortly after fertilization and prior to fusion. The major portion of DNA synthesis, as evidenced by thymidine uptake, appears to be during a 20 to 30 minute period after fertilization. Fertilization is associated with the activation of a mechanism which initiates early and independent replication of DNA in both the male and female pronuclei.

OBSERVATIONS ON THE UPTAKE OF TRITIATED THYMIDINE IN THE PRONUCLEI OF FERTILIZED SAND DOLLAR EMBRYOS

PubMed Central

Simmel, Eva B.; Karnofsky, David A.

1961-01-01

Following fertilization of the egg of the sand dollar Echinarachnius parma, tritiated thymidine (H3TDR) was taken up independently by the male and female pronuclei beginning within about 15 to 20 minutes, and the labeled pronuclei fused at about 30 to 40 minutes. At cleavage 90 minutes later the labeled nuclear material was distributed to both daughter cells. Unfertilized eggs and sperm exposed to H3TDR did not show nuclear localization of thymidine. DNA replication, thus, is initiated in the haploid pronuclei shortly after fertilization and prior to fusion. The major portion of DNA synthesis, as evidenced by thymidine uptake, appears to be during a 20 to 30 minute period after fertilization. Fertilization is associated with the activation of a mechanism which initiates early and independent replication of DNA in both the male and female pronuclei. PMID:19866590

Interplay of protein and DNA structure revealed in simulations of the lac operon.

PubMed

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

PubMed

Bogacheva, Mariia; Egorova, Anna; Slita, Anna; Maretina, Marianna; Baranov, Vladislav; Kiselev, Anton

2017-11-01

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

PubMed

Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

2016-05-25

Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).

Heparin affinity purification of extracellular vesicles

PubMed Central

Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

2015-01-01

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

High-Throughput Identification of Combinatorial Ligands for DNA Delivery in Cell Culture

NASA Astrophysics Data System (ADS)

Svahn, Mathias G.; Rabe, Kersten S.; Barger, Geoffrey; EL-Andaloussi, Samir; Simonson, Oscar E.; Didier, Boturyn; Olivier, Renaudet; Dumy, Pascal; Brandén, Lars J.; Niemeyer, Christof M.; Smith, C. I. Edvard

2008-10-01

Finding the optimal combinations of ligands for tissue-specific delivery is tedious even if only a few well-established compounds are tested. The cargo affects the receptor-ligand interaction, especially when it is charged like DNA. The ligand should therefore be evaluated together with its cargo. Several viruses have been shown to interact with more than one receptor, for efficient internalization. We here present a DNA oligonucleotide-based method for inexpensive and rapid screening of biotin labeled ligands for combinatorial effects on cellular binding and uptake. The oligonucleotide complex was designed as a 44 bp double-stranded DNA oligonucleotide with one central streptavidin molecule and a second streptavidin at the terminus. The use of a highly advanced robotic platform ensured stringent processing and execution of the experiments. The oligonucleotides were fluorescently labeled and used for detection and analysis of cell-bound, internalized and intra-cellular compartmentalized constructs by an automated line-scanning confocal microscope, IN Cell Analyzer 3000. All possible combinations of 22 ligands were explored in sets of 2 and tested on 6 different human cell lines in triplicates. In total, 10 000 transfections were performed on the automation platform. Cell-specific combinations of ligands were identified and their relative position on the scaffold oligonucleotide was found to be of importance. The ligands were found to be cargo dependent, carbohydrates were more potent for DNA delivery whereas cell penetrating peptides were more potent for delivery of less charged particles.

Functional Analysis of the ComK Protein of Bacillus coagulans

PubMed Central

Kovács, Ákos T.; Eckhardt, Tom H.; van Kranenburg, Richard; Kuipers, Oscar P.

2013-01-01

The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis- and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNA-uptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B. subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis. PMID:23301076

Plasma membrane translocation of a protein needle based on a triple-stranded β-helix motif.

PubMed

Sanghamitra, Nusrat J M; Inaba, Hiroshi; Arisaka, Fumio; Ohtan Wang, Dan; Kanamaru, Shuji; Kitagawa, Susumu; Ueno, Takafumi

2014-10-01

Plasma membrane translocation is challenging due to the barrier of the cell membrane. Contrary to the synthetic cell-penetrating materials, tailed bacteriophages use cell-puncturing protein needles to puncture the cell membranes as an initial step of the DNA injection process. Cell-puncturing protein needles are thought to remain functional in the native phages. In this paper, we found that a bacteriophage T4 derived protein needle of 16 nm length spontaneously translocates through the living cell membrane. The β-helical protein needle (β-PN) internalizes into human red blood cells that lack endocytic machinery. By comparing the cellular uptake of β-PNs with modified surface charge, it is shown that the uptake efficiency is maximum when it has a negative charge corresponding to a zeta potential value of -16 mV. In HeLa cells, uptake of β-PN incorporates endocytosis independent mechanisms with partial macropinocytosis dependence. The endocytosis dependence of the uptake increases when the surface charges of β-PNs are modified to positive or negative. Thus, these results suggest that natural DNA injecting machinery can serve as an inspiration to design new class of cell-penetrating materials with a tailored mechanism.

Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer

PubMed Central

Church, Timothy Robert; Wandell, Michael; Lofton-Day, Catherine; Mongin, Steven J; Burger, Matthias; Payne, Shannon R; Castaños-Vélez, Esmeralda; Blumenstein, Brent A; Rösch, Thomas; Osborn, Neal; Snover, Dale; Day, Robert W; Ransohoff, David F

2014-01-01

Background As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase both, but none has yet been tested in a screening setting. Objective We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population. Design Asymptomatic individuals ≥50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates. Results 7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude rate 50.9%); for CRC stages I–IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%). Conclusions Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas. Clinical Trial Registration Number: NCT00855348 PMID:23408352

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1988-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

PubMed Central

Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

1987-01-01

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. Images PMID:2823261

THE ATM-SMC1 PATHWAY IS ESSENTIAL FOR ACTIVATION OF THE CHROMIUM[VI]-INDUCED S-PHASE CHECKPOINT

EPA Science Inventory

Hexavalent chromium (Cr[VI] is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisims by which mammalian cells respond to Cr-induced DNA damage remain to b...

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

PubMed

Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

2016-09-01

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Non-hyperfunctioning nodules from multinodular goiters: a minor role in pathogenesis for somatic activating mutations in the TSH-receptor and Gsalpha subunit genes.

PubMed

Derrien, C; Sonnet, E; Gicquel, I; Le Gall, J Y; Poirier, J Y; David, V; Maugendre, D

2001-05-01

Constitutive activation of the cAMP pathway stimulates thyrocyte proliferation. Gain-of-function mutations in Gsalpha protein have already been identified in thyroid nodules which have lost the ability to trap iodine. In contrast, most of the studies failed to detect somatic activating mutations in the thyrotropin receptor (TSH-R) in non-hyperfunctioning thyroid tumors. The aim of this study was to screen for mutations TSH-R exon 10, encoding the whole intracytoplasmic area involved in signal transduction, and Gsalpha exons 8 and 9, containing the two hot-spot codons 201 and 227, in a subset of non-hyperfunctioning nodules from multinodular goiter. Identified by matching ultrasonography and scintiscan, 22 eufunctioning (normal 99Tc uptake) and 15 nonfunctioning (decreased 99Tc uptake) nodules from 27 non-toxic multinodular goiters were isolated. After DNA extraction, TSH-R exon 10 was analyzed by direct sequencing of the PCR products and Gsalpha exons 8 and 9 by Denaturing Gradient Gel Electrophoresis. No mutation of TSH-R or Gsalpha was detected in the 37 nodules analyzed. This absence of mutation, despite the use of two sensitive screening methods associated with the analysis of the TSH-R whole intracytoplasmic area and Gsalpha two hot-spot codons, suggests that TSH-R and Gsalpha play a minor role in the pathogenesis of non-toxic nodules from multinodular goiters.

[Pathogenesis, diagnosis and therapy of Legionella infections].

PubMed

Lück, P C; Steinert, M

2006-05-01

Legionella species are ubiquitous in aquatic environments. About 50 years ago they entered the engineered (technical) environment, i.e. warm water systems with zones of stagnation. Since that time they represent a hygienic problem. After transmission to humans via aerosols legionellae might cause Legionella pneumonia (legionnaires' disease) or influenza-like respiratory infections (Pontiac fever). Epidemiological data suggest that Legionella strains might differ substantially in their virulence properties. Although the molecular basis is not understood L. pneumophila serogroup 1 especially MAb 3/1-positive strains cause the majority of infections. The main virulence feature is the ability to multiply intracellularly. After uptake into macrophages legionellae multiply in a specialized vacuole and finally lyse their host cells. Several bacterial factors like surface components, secretion systems and iron uptake systems are involved in this process. Since the clinical picture of Legionella pneumonia does not allow differentiation from pneumoniae caused by other pathogens, microbiological diagnostic methods are needed to establish the diagnosis. Cultivation of legionellae from clinical specimens, detection of antigens and DNA in patients' samples and detection of antibodies in serum samples are suitable methods. However, none of the diagnostic tests presently available offers the desired quality with respect to sensitivity and specificity. Therefore, the standard technique is to use several diagnostic tests in parallel. Advantages and disadvantages of the diagnostic procedures are discussed. Therapeutic options for Legionella infections are newer macrolides like azithromycin and chinolones (ciprofloxacin, levofloxacin and moxifloxacin).

Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA

PubMed Central

Prevette, Lisa E.; Mullen, Douglas G.; Banaszak Holl, Mark M.

2010-01-01

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression. PMID:20349965

Moisture-induced aggregation of lyophilized DNA and its prevention.

PubMed

Sharma, Vikas K; Klibanov, Alexander M

2007-01-01

To investigate the moisture-induced aggregation (i.e., a loss of solubility in water) of DNA in a solid state and to develop rational strategies for its prevention. Lyophilized calf thymus DNA was exposed to relative humidity (RH) levels from 11% to 96% at 55 degrees C. Following a 24-h incubation under these stressed conditions, the solubility of DNA in different aqueous solutions and the water uptake of DNA were determined. The effects of solution pH and NaCl concentration and the presence of excipients (dextran and sucrose) on the subsequent moisture-induced aggregation of DNA were examined. The extent of this aggregation was compared with that of a supercoiled plasmid DNA. Upon a 24-h incubation at 55 degrees C, calf thymus DNA underwent a major moisture-induced aggregation reaching a maximum at a 60% RH; in contrast, the single-stranded DNA exhibited the maximal aggregation at a 96% RH. Moisture uptake and aqueous solubility studies revealed that the aggregation was primarily due to formation of inter-strand hydrogen bonds. Aggregation of DNA also proceeded at 37 degrees C, albeit at a slower rate. Solution pH and NaCl concentration affected DNA aggregation only at higher RH levels. This aggregation was markedly reduced by co-lyophilization with dextran or sucrose (but not with PEG). The aggregation pattern of a supercoiled plasmid DNA was similar to that of its linear calf thymus counterpart. The moisture-induced aggregation of lyophilized DNA is caused mainly by non-covalent cross-links between disordered, single-stranded regions of DNA. At high RH levels, renaturation and aggregation of DNA compete with each other. The aggregation is minimized at low RH levels, at optimal solution pH and salt concentration prior to lyophilization, and by co-lyophilizing with excipients capable of forming multiple hydrogen bonds, e.g., dextran and sucrose.

Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats.

PubMed

Okayama, Takashige; Yoshisue, Kunihiro; Kuwata, Keizo; Komuro, Masahito; Ohta, Shigeru; Nagayama, Sekio

2012-02-01

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

Relationship between pretreatment level of plasma Epstein-Barr virus DNA, tumor burden, and metabolic activity in advanced nasopharyngeal carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Brigette; King, Ann; Lo, Y.M. Dennis

Purpose: Plasma Epstein-Barr virus DNA (pEBV DNA) is an important prognostic marker in nasopharyngeal carcinoma (NPC). This study tested the hypotheses that pEBV DNA reflects tumor burden and metabolic activity by evaluating its relationship with tumor volume and {sup 18}F-fluorodeoxyglucose ({sup 18}F-FDG) uptake in NPC. Methods and Materials: Pre-treatment pEBV DNA analysis, {sup 18}F-FDG positron emission tomography-computed tomography scan (PET-CT) and magnetic resonance imaging (MRI) of the head and neck were performed in 57 patients. Net volume (cm{sup 3}) of the primary tumor (T{sub vol}) and regional nodes (N{sub vol}) were quantified on MRI. {sup 18}F-FDG uptake was expressed asmore » the maximum standardized uptake value (SUV{sub max}) at the primary tumor (T{sub suv}) and regional nodes (N{sub suv}). Lesions with SUV{sub max} {>=} 2.5 were considered malignant. Relationship between SUV{sub max}, natural logarithm (log) of pEBV DNA, and square root (sq) of MRI volumes was analyzed using the Wilcoxon test. A linear regression model was constructed to test for any interaction between variables and disease stage. Results: Log-pEBV DNA showed significant correlation with sq-T{sub vol} (r = 0.393), sq-N{sub vol} (r = 0.452), total tumor volume (sq-Total{sub vol} = T{sub vol} + N{sub vol}, r = 0.554), T{sub suv} (r = 0.276), N{sub suv} (r = 0.434), and total SUV{sub max} (Total{sub suv} = T{sub suv} + N{sub suv}, r = 0.457). Likewise, sq-T{sub vol} was correlated to T{sub suv} (r 0.426), and sq-N{sub vol} with N{sub suv} (r = 0.651). Regression analysis showed that only log-pEBV DNA was significantly associated with sq-Total{sub vol} (p < 0.001; parameter estimate = 8.844; 95% confidence interval = 3.986-13.703), whereas Sq-T{sub vol} was significantly associated with T{sub suv} (p = 0.002; parameter estimate = 3.923; 95% confidence interval = 1.498-6.348). Conclusion: This study supports the hypothesis that cell-free plasma EBV DNA is a marker of tumor burden in EBV-related NPC.« less

Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation

USGS Publications Warehouse

Walker, Donald M.; Leys, Jacob E.; Dunham, Kelly E.; Oliver, Joshua C.; Schiller, Emily E.; Stephenson, Kelsey S.; Kimrey, John T.; Wooten, Jessica; Rogers, Mark W.

2017-01-01

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.

Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation.

PubMed

Walker, Donald M; Leys, Jacob E; Dunham, Kelly E; Oliver, Joshua C; Schiller, Emily E; Stephenson, Kelsey S; Kimrey, John T; Wooten, Jessica; Rogers, Mark W

2017-11-01

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations. © 2017 John Wiley & Sons Ltd.

Abnormal brain aging as a radical-related disease: A new target for nuclear medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujibayashi, Y.; Yamamoto, S.; Waki, A.

DNA damages caused by endogenously produced radicals are closely correlated with aging. Among them, mitochondrial DNA (mtDNA) deletions have been reported as a memory of DNA damage by oxygen radicals. In fact, clinical as well as experimental studies indicated the accumulation of deleted mtDNA in the brain, myocardium and son on, in aged subjects. In our previous work, radioiodinated radical trapping agent, p-iodophenyl-N-t-butylnitrone, and hypoxia imaging agent, Cu-62 diacetyl-bis-N-4-methyl-thiosemicarbazone have been developed for the diagnosis of radical-related diseases, such as ischemic, inflammation, cancer or aging. The aim of the present work was to evaluate these agents for brain aging studies.more » In our university, an unique animal model, a senescence accelerated model mouse (SAM), has been established. Among the various substrains, SAMP8 showing memory deterioration in its young age ({approximately}3 month) was basically evaluated as an abnormal brain aging model with mtDNA deletion. As controls, SAMR1 showing normal aging and ddY mice were used. MtDNA deletion n the brain was analyzed with polymerase-chain reaction (PCR) method, and relationship between mtDNA deletion and brain uptake of IPBN or Cu-62-ATSM was studied. In 1-3 month old SAMP8 brain, multiple mtDNa deletions were already found and their content was significantly higher than that of SAMR1 or age-matched ddY control. Thus, it was cleared that SAMP8 brain has high tendency to be attacked by endogenously produced oxygen radicals, possibly from its birth. Both IPBN and Cu-ATSM showed significantly higher accumulation in the SAMP8 brain than in the SAMR1 brain, indicating that these agents have high possibility for the early detection of abnormal brain aging as a radical-related disease.« less

Linking loss of sodium-iodide symporter expression to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyckesvärd, Madeleine Nordén; Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg; Kapoor, Nirmal

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression andmore » transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.« less

Enzyme-synthesized Poly(amine-co-esters) as Non-viral Vectors for Gene Delivery

PubMed Central

Liu, Jie; Jiang, Zhaozhong; Zhou, Jiangbing; Zhang, Shengmin; Saltzman, W. Mark

2010-01-01

A family of biodegradable poly(amine-co-esters) was synthesized in one step via enzymatic copolymerization of diesters with amino-substituted diols. Diesters of length C4–C12 (i.e., from succinate to dodecanedioate) were successfully copolymerized with diethanolamines with either an alkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituent on the nitrogen. Upon protonation at slightly acidic conditions, these poly(amine-co-esters) readily turned to cationic polyelectrolytes, which were capable of condensing with polyanionic DNA to form nanometer-sized polyplexes. In vitro screening with pLucDNA revealed that two of the copolymers, poly(N-methyldiethyleneamine sebacate) (PMSC) and poly(N-ethyldiethyleneamine sebacate) (PESC), possessed comparable or higher transfection efficiencies compared to Lipofectamine 2000. PMSC/pLucDNA and PESC/pLucDNA nanoparticles had desirable particle sizes (40–70 nm) for cellular uptake and were capable of functioning as proton sponges to facilitate endosomal escape after cellular uptake. These polyplex nanoparticles exhibited extremely low cytotoxicity. Furthermore, in vivo gene transfection experiments revealed that PMSC is a substantially more effective gene carrier than PEI in delivering pLucDNAto cells in tumors in mice. All these properties suggest that poly(amine-co-esters) are promising non-viral vectors for safe and efficient DNA delivery in gene therapy. PMID:21171165

Calcium-dependent mitochondrial formation of species mediating DNA single strand breakage in U937 cells exposed to sublethal concentrations of tert-butylhydroperoxide.

PubMed

Guidarelli, A; Clementi, E; Sciorati, C; Cattabeni, F; Cantoni, O

1997-10-01

Treatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca++ from the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca++ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H2O2. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl2. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid. The mechanism(s) whereby Ca++ promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species.

Preparation of astaxanthin-loaded DNA/chitosan nanoparticles for improved cellular uptake and antioxidation capability.

PubMed

Wang, Qian; Zhao, Yingyuan; Guan, Lei; Zhang, Yaping; Dang, Qifeng; Dong, Ping; Li, Jing; Liang, Xingguo

2017-07-15

DNA/chitosan co-assemblies were initially used as nanocarriers for efficient astaxanthin encapsulation and delivery. The obtained astaxanthin-loaded DNA/chitosan (ADC) colloidal system was transparent and homogenous, with astaxanthin content up to 65μg/ml. Compared to free astaxanthin, ADC nanoparticles with an astaxanthin concentration as low as 3.35nM still showed a more powerful cytoprotective effect on H 2 O 2 -induced oxidative cell damage, and improved cell viability from 49.9% to 61.9%. The ROS scavenging efficiency of ADC nanoparticles was as high as 54.3%, which was 2-fold higher than that of free astaxanthin. Besides this, ADC nanoparticles were easily engulfed by Caco-2 cells in a short time, indicating that the encapsulated astaxanthin could be absorbed through endocytosis by intestinal epithelial cells. The improved antioxidation capability and facilitated cellular uptake enabled the ADC nanoparticles to be good candidates for efficient delivery and absorption of astaxanthin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

NASA Astrophysics Data System (ADS)

Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

2015-07-01

Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

Detection of viral infection and gene expression in clinical tissue specimens using branched DNA (bDNA) in situ hybridization.

PubMed

Kenny, Daryn; Shen, Lu-Ping; Kolberg, Janice A

2002-09-01

In situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.

Double labeling of human leukemic cells using /sup 3/H-cytarabine and monoclonal antibody against bromodeoxyuridine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, A.; Preisler, H.D.

A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of /sup 3/H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to /sup 3/H-cytarabine. Other radioactively labeled probes can be used as well.

Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery.

PubMed

Chowdhury, E H; Akaike, Toshihiro

2005-05-20

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy. (c) 2005 Wiley Periodicals, Inc.

Microbiology and Moisture Uptake of Desert Soils

NASA Astrophysics Data System (ADS)

Kress, M. E.; Bryant, E. P.; Morgan, S. W.; Rech, S.; McKay, C. P.

2005-12-01

We have initiated an interdisciplinary study of the microbiology and water content of desert soils to better understand microbial activity in extreme arid environments. Water is the one constituent that no organism can live without; nevertheless, there are places on Earth with an annual rainfall near zero that do support microbial ecosystems. These hyperarid deserts (e.g. Atacama and the Antarctic Dry Valleys) are the closest terrestrial analogs to Mars, which is the subject of future exploration motivated by the search for life beyond Earth. We are modeling the moisture uptake by soils in hyperarid environments to quantify the environmental constraints that regulate the survival and growth of micro-organisms. Together with the studies of moisture uptake, we are also characterizing the microbial population in these soils using molecular and culturing methods. We are in the process of extracting DNA from these soils using MoBio extraction kits. This DNA will be used as a template to amplify bacterial and eukaryotic ribosomal DNA to determine the diversity of the microbial population. We also have been attempting to determine the density of organisms by culturing on one-half strength R2A agar. The long-range goal of this research is to identify special adaptations of terrestrial life that allow them to inhabit extreme arid environments, while simultaneously quantifying the environmental parameters that enforce limits on these organisms' growth and survival.

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

PubMed

Player, A N; Shen, L P; Kenny, D; Antao, V P; Kolberg, J A

2001-05-01

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles.

PubMed

Davy, Christina M; Kidd, Anne G; Wilson, Chris C

2015-01-01

Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2-10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species.

Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles

PubMed Central

Davy, Christina M.; Kidd, Anne G.; Wilson, Chris C.

2015-01-01

Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2–10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species. PMID:26200348

Methods of DNA methylation detection

NASA Technical Reports Server (NTRS)

Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

2010-01-01

The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

PubMed Central

Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

2002-01-01

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells.

PubMed

Ferreira, Martiña; Blanco, Lucía; Garrido, Alejandro; Vieites, Juan M; Cabado, Ana G

2013-05-01

The toxic effects of the organotin compounds (OTCs) monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were evaluated in vitro in a neuroblastoma human cell line. Mechanisms of cell death, apoptosis versus necrosis, were studied by using several markers: inhibition of cell viability and proliferation, F-actin, and mitochondrial membrane potential changes as well as reactive oxygen species (ROS) production and DNA fragmentation. The most toxic effects were detected with DBT and TBT even at very low concentrations (0.1-1 μM). In contrast, MBT induced lighter cytotoxic changes at the higher doses tested. None of the studied compounds stimulated propidium iodide uptake, although the most toxic chemical, TBT, caused lactate dehydrogenase release at the higher concentrations tested. These findings suggest that in neuroblastoma, OTC-induced cytotoxicity involves different pathways depending on the compound, concentration, and incubation time. A screening method for DBT and TBT quantification based on cell viability loss was developed, allowing a fast detection alternative to complex methodology.

Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography.

PubMed

Lolas, Ihab Bishara; Chen, Xijuan; Bester, Kai; Nielsen, Jeppe Lund

2012-11-01

Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants.

Carbon nanotubes as gene carriers: Focus on internalization pathways related to functionalization and properties.

PubMed

Caoduro, Cécile; Hervouet, Eric; Girard-Thernier, Corine; Gharbi, Tijani; Boulahdour, Hatem; Delage-Mourroux, Régis; Pudlo, Marc

2017-02-01

Carbon nanotubes represent promising transporters for delivery of DNA and other biomolecules into living cells. Various methods of CNTs surface functionalization have been developed. These are essential to improve CNTs dispersibility and permit their interactions with biological structures that broaden their use in advanced biomedical applications. The present review discusses the different single walled carbon nanotubes and multiwalled carbon nanotubes functionalization methods, leading to the formation of optimized and functionalized-CNT complexes with DNA. F-CNTs are recognized as efficient and promising gene carriers. Emphasis is then placed on the processes used by f-CNTs/DNA complexes to cross cell membranes. Energy independent pathways and uptake mechanisms dependent on energy, such as endocytosis or phagocytosis, are reported by many studies, and if these mechanisms seem contradictory at first sight, a detailed review of the literature illustrates that they are rather complementary. Preferential use of one or the other depends on the DNA and CNTs chemical nature and physical parameters, experimental procedures and cell types. Efficient non-viral gene delivery is desirable, yet challenging. CNTs appear as a promising solution to penetrate into cells and successfully deliver DNA. Moreover, the field of use of CNTs as gene carrier is large and is currently growing. This critical review summarizes the development and evaluation of CNTs as intracellular gene delivery system and provides an overview of functionalized CNTs/DNA cellular uptake mechanisms, depending on several parameters of CNTs/DNA complexes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

PubMed

Lee, Jyun-Lin; Lo, Chia-Wen; Ka, Shuk-Man; Chen, Ann; Chen, Wen-Shiang

2012-05-30

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

PubMed Central

Sun, Xun; Chen, Simu; Han, Jianfeng; Zhang, Zhirong

2012-01-01

Background To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs) more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG) and a series of its mannosylated derivatives. Methods PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs) using flow cytometry. Results PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation. Conclusion These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system. PMID:22745554

In Vitro Cellular Gene Delivery Employing a Novel Composite Material of Single-Walled Carbon Nanotubes Associated With Designed Peptides With Pegylation.

PubMed

Ohta, Takahisa; Hashida, Yasuhiko; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

2017-03-01

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical fields including application for drug delivery system. In this study, we developed a novel gene delivery system employing SWCNTs associated with polycationic and amphiphilic H-(-Lys-Trp-Lys-Gly-) 7 -OH [(KWKG) 7 ] peptides having pegylation. SWCNTs wrapped with (KWKG) 7 formed a complex with plasmid DNA (pDNA) in aqueous solution based on polyionic interaction but later underwent aggregation. On the other hand, a complex of pDNA and SWCNT-(KWKG) 7 modified with polyethylene glycol (PEG) chains of 12 units [SWCNT-(KWKG) 7 -(PEG) 12 ] afforded good dispersion stability for 24 h even in a cell culture medium. The in vitro cellular uptake of SWCNT-(KWKG) 7 -(PEG) 12 /pDNA complex prepared with fluorescence-labeled pDNA was evaluated with fluorescent microscopic observation and flow cytometry. The uptake by A549 human lung adenocarcinoma epithelial cells increased along with the extent of pegylation, suggesting the importance of dispersion stability in addition to the cationic charge which facilitates ionic cellular interaction. The expression of pDNA encoding the monomeric Kusabira-Orange 2 fluorescent protein in the form of the SWCNT-(KWKG) 7 -(PEG) 12 /pDNA complex demonstrated remarkable enhancement of transfection depending also on the extent of pegylation and the N/P ratio. The potential of the SWCNT composite wrapped with polycationic and amphiphilic (KWKG) 7 with pegylation as a carrier for gene delivery was demonstrated. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Detection of bladder metabolic artifacts in (18)F-FDG PET imaging.

PubMed

Roman-Jimenez, Geoffrey; Crevoisier, Renaud De; Leseur, Julie; Devillers, Anne; Ospina, Juan David; Simon, Antoine; Terve, Pierre; Acosta, Oscar

2016-04-01

Positron emission tomography using (18)F-fluorodeoxyglucose ((18)F-FDG-PET) is a widely used imaging modality in oncology. It enables significant functional information to be included in analyses of anatomical data provided by other image modalities. Although PET offers high sensitivity in detecting suspected malignant metabolism, (18)F-FDG uptake is not tumor-specific and can also be fixed in surrounding healthy tissue, which may consequently be mistaken as cancerous. PET analyses may be particularly hampered in pelvic-located cancers by the bladder׳s physiological uptake potentially obliterating the tumor uptake. In this paper, we propose a novel method for detecting (18)F-FDG bladder artifacts based on a multi-feature double-step classification approach. Using two manually defined seeds (tumor and bladder), the method consists of a semi-automated double-step clustering strategy that simultaneously takes into consideration standard uptake values (SUV) on PET, Hounsfield values on computed tomography (CT), and the distance to the seeds. This method was performed on 52 PET/CT images from patients treated for locally advanced cervical cancer. Manual delineations of the bladder on CT images were used in order to evaluate bladder uptake detection capability. Tumor preservation was evaluated using a manual segmentation of the tumor, with a threshold of 42% of the maximal uptake within the tumor. Robustness was assessed by randomly selecting different initial seeds. The classification averages were 0.94±0.09 for sensitivity, 0.98±0.01 specificity, and 0.98±0.01 accuracy. These results suggest that this method is able to detect most (18)F-FDG bladder metabolism artifacts while preserving tumor uptake, and could thus be used as a pre-processing step for further non-parasitized PET analyses. Copyright © 2016. Published by Elsevier Ltd.

A cell-targeted, size-photocontrollable, nuclear-uptake nanodrug delivery system for drug-resistant cancer therapy.

PubMed

Qiu, Liping; Chen, Tao; Öçsoy, Ismail; Yasun, Emir; Wu, Cuichen; Zhu, Guizhi; You, Mingxu; Han, Da; Jiang, Jianhui; Yu, Ruqin; Tan, Weihong

2015-01-14

The development of multidrug resistance (MDR) has become an increasingly serious problem in cancer therapy. The cell-membrane overexpression of P-glycoprotein (P-gp), which can actively efflux various anticancer drugs from the cell, is a major mechanism of MDR. Nuclear-uptake nanodrug delivery systems, which enable intranuclear release of anticancer drugs, are expected to address this challenge by bypassing P-gp. However, before entering the nucleus, the nanocarrier must pass through the cell membrane, necessitating coordination between intracellular and intranuclear delivery. To accommodate this requirement, we have used DNA self-assembly to develop a nuclear-uptake nanodrug system carried by a cell-targeted near-infrared (NIR)-responsive nanotruck for drug-resistant cancer therapy. Via DNA hybridization, small drug-loaded gold nanoparticles (termed nanodrugs) can self-assemble onto the side face of a silver-gold nanorod (NR, termed nanotruck) whose end faces were modified with a cell type-specific internalizing aptamer. By using this size-photocontrollable nanodrug delivery system, anticancer drugs can be efficiently accumulated in the nuclei to effectively kill the cancer cells.

A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

PubMed

Wei, Wei; Gao, Chunyan; Xiong, Yanxiang; Zhang, Yuanjian; Liu, Songqin; Pu, Yuepu

2015-01-01

DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate. Copyright © 2014 Elsevier B.V. All rights reserved.

Biomimetic nanochannels based biosensor for ultrasensitive and label-free detection of nucleic acids.

PubMed

Sun, Zhongyue; Liao, Tangbin; Zhang, Yulin; Shu, Jing; Zhang, Hong; Zhang, Guo-Jun

2016-12-15

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Reconstruction of Toll-like receptor 9-mediated responses in HEK-Blue hTLR9 cells by transfection of human macrophage scavenger receptor 1 gene.

PubMed

Ohtsuki, Shozo; Takahashi, Yuki; Inoue, Takao; Takakura, Yoshinobu; Nishikawa, Makiya

2017-10-20

We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

DOE PAGES

Wang, Sisi; Zhang, Hongyong; Scharadin, Tiffany M.; ...

2016-01-22

Here, we report the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formationmore » and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.« less

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Sisi; Zhang, Hongyong; Scharadin, Tiffany M.

Here, we report the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formationmore » and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.« less

Fate and Uptake of Pharmaceuticals in Soil–Plant Systems

PubMed Central

2014-01-01

Pharmaceuticals have been detected in the soil environment where there is the potential for uptake into crops. This study explored the fate and uptake of pharmaceuticals (carbamazepine, diclofenac, fluoxetine, propranolol, sulfamethazine) and a personal care product (triclosan) in soil–plant systems using radish (Raphanus sativus) and ryegrass (Lolium perenne). Five of the six chemicals were detected in plant tissue. Carbamazepine was taken up to the greatest extent in both the radish (52 μg/g) and ryegrass (33 μg/g), whereas sulfamethazine uptake was below the limit of quantitation (LOQ) (<0.01 μg/g). In the soil, concentrations of diclofenac and sulfamethazine dropped below the LOQ after 7 days. However, all pharmaceuticals were still detectable in the pore water at the end of the experiment. The results demonstrate the ability of plant species to accumulate pharmaceuticals from soils with uptake apparently specific to both plant species and chemical. Results can be partly explained by the hydrophobicity and extent of ionization of each chemical in the soil. PMID:24405013

Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

PubMed

Shigeta, S; Toyoshima, M; Kitatani, K; Ishibashi, M; Usui, T; Yaegashi, N

2016-07-07

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Assessing environmental DNA detection in controlled lentic systems.

PubMed

Moyer, Gregory R; Díaz-Ferguson, Edgardo; Hill, Jeffrey E; Shea, Colin

2014-01-01

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m3). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3-5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42-73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m3 or 1.75 g/m3).

Transport Distance of Invertebrate Environmental DNA in a Natural River

PubMed Central

Deiner, Kristy; Altermatt, Florian

2014-01-01

Environmental DNA (eDNA) monitoring is a novel molecular technique to detect species in natural habitats. Many eDNA studies in aquatic systems have focused on lake or ponds, and/or on large vertebrate species, but applications to invertebrates in river systems are emerging. A challenge in applying eDNA monitoring in flowing waters is that a species' DNA can be transported downstream. Whether and how far eDNA can be detected due to downstream transport remains largely unknown. In this study we tested for downstream detection of eDNA for two invertebrate species, Daphnia longispina and Unio tumidus, which are lake dwelling species in our study area. The goal was to determine how far away from the source population in a lake their eDNA could be detected in an outflowing river. We sampled water from eleven river sites in regular intervals up to 12.3 km downstream of the lake, developed new eDNA probes for both species, and used a standard PCR and Sanger sequencing detection method to confirm presence of each species' eDNA in the river. We detected D. longispina at all locations and across two time points (July and October); whereas with U. tumidus, we observed a decreased detection rate and did not detect its eDNA after 9.1 km. We also observed a difference in detection for this species at different times of year. The observed movement of eDNA from the source amounting to nearly 10 km for these species indicates that the resolution of an eDNA sample can be large in river systems. Our results indicate that there may be species' specific transport distances for eDNA and demonstrate for the first time that invertebrate eDNA can persist over relatively large distances in a natural river system. PMID:24523940

Direct detection of methylation in genomic DNA

PubMed Central

Bart, A.; van Passel, M. W. J.; van Amsterdam, K.; van der Ende, A.

2005-01-01

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote. PMID:16091626

Potential of environmental DNA to evaluate Northern pike (Esox lucius) eradication efforts: An experimental test and case study

USGS Publications Warehouse

Dunker, Kristine J.; Sepulveda, Adam; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

PubMed

Dunker, Kristine J; Sepulveda, Adam J; Massengill, Robert L; Olsen, Jeffrey B; Russ, Ora L; Wenburg, John K; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study

PubMed Central

Dunker, Kristine J.; Sepulveda, Adam J.; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water. PMID:27626271

Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons

PubMed Central

Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

2015-01-01

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models. PMID:25874630

Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive Burmese pythons

USGS Publications Warehouse

Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

2015-01-01

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.

Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive burmese pythons.

PubMed

Hunter, Margaret E; Oyler-McCance, Sara J; Dorazio, Robert M; Fike, Jennifer A; Smith, Brian J; Hunter, Charles T; Reed, Robert N; Hart, Kristen M

2015-01-01

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.

An evaluation of the efficacy of using environmental DNA (eDNA) to detect giant gartersnakes (Thamnophis gigas)

USGS Publications Warehouse

Halstead, Brian J.; Wood, Dustin A.; Bowen, Lizabeth; Waters, Shannon C.; Vandergast, Amy G.; Ersan, Julia S.; Skalos, Shannon M.; Casazza, Michael L.

2017-09-28

Detecting populations of rare or cryptic species is essential for their conservation. For species like giant gartersnakes (Thamnophis gigas), conventional survey methods can be expensive and inefficient. These sampling difficulties might be overcome by modern techniques that detect deoxyribonucleic acid (DNA) shed by organisms into the environment (eDNA). We evaluated the efficacy of detecting giant gartersnake eDNA in water samples from the laboratory and at locations with known giant gartersnake populations in the Sacramento Valley of California, and failed to detect giant gartersnake DNA in most laboratory and all field samples. Aspects of giant gartersnake biology—such as highly keratinized skin and spending extensive time in the terrestrial environment, as well as hot, sunny, and turbid conditions in wetlands and canals of the Sacramento Valley—likely contributed to low detection probabilities. Although detection of eDNA shows promise under many conditions, further development is needed before sampling for eDNA is a viable option for detecting giant gartersnake populations.

DNA Clutch Probes for Circulating Tumor DNA Analysis.

PubMed

Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

2016-08-31

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

Molecular identification and functional characteristics of peptide transporters in the bonnethead shark (Sphyrna tiburo).

PubMed

Hart, Hannah R; Evans, Andrew N; Gelsleichter, James; Ahearn, Gregory A

2016-10-01

Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark (Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H(+)-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies.

A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.

PubMed

Mu, Di; Yan, Liang; Tang, Hui; Liao, Yong

2015-10-01

To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients. A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples. The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.

[A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].

PubMed

Wang, Mei-Rong; Qiu, Ning; Lu, Shi-Chun; Xiu, Dian-Rong; Yu, Jian-Guo; Li, Tong; Liu, Xue-En; Zhuang, Hui

2011-05-01

To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue. Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%. A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

Fluorescent carbon nanoparticle-based lateral flow biosensor for ultrasensitive detection of DNA.

PubMed

Takalkar, Sunitha; Baryeh, Kwaku; Liu, Guodong

2017-12-15

We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of the global ocean sampling (GOS) project for trends in iron uptake by surface ocean microbes.

PubMed

Toulza, Eve; Tagliabue, Alessandro; Blain, Stéphane; Piganeau, Gwenael

2012-01-01

Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe(2+) versus Fe(3+) uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China

PubMed Central

Zhou, Rui; Jin, Qi; Fan, Yang; Bei, Weicheng; Chen, Huanchun

2011-01-01

Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis. PMID:21611187

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

I Environmental DNA sampling is more sensitive than a traditional survey technique for detecting an aquatic invader.

PubMed

Smart, Adam S; Tingley, Reid; Weeks, Andrew R; van Rooyen, Anthony R; McCarthy, Michael A

2015-10-01

Effective management of alien species requires detecting populations in the early stages of invasion. Environmental DNA (eDNA) sampling can detect aquatic species at relatively low densities, but few studies have directly compared detection probabilities of eDNA sampling with those of traditional sampling methods. We compare the ability of a traditional sampling technique (bottle trapping) and eDNA to detect a recently established invader, the smooth newt Lissotriton vulgaris vulgaris, at seven field sites in Melbourne, Australia. Over a four-month period, per-trap detection probabilities ranged from 0.01 to 0.26 among sites where L. v. vulgaris was detected, whereas per-sample eDNA estimates were much higher (0.29-1.0). Detection probabilities of both methods varied temporally (across days and months), but temporal variation appeared to be uncorrelated between methods. Only estimates of spatial variation were strongly correlated across the two sampling techniques. Environmental variables (water depth, rainfall, ambient temperature) were not clearly correlated with detection probabilities estimated via trapping, whereas eDNA detection probabilities were negatively correlated with water depth, possibly reflecting higher eDNA concentrations at lower water levels. Our findings demonstrate that eDNA sampling can be an order of magnitude more sensitive than traditional methods, and illustrate that traditional- and eDNA-based surveys can provide independent information on species distributions when occupancy surveys are conducted over short timescales.

DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

PubMed

Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

2013-02-01

Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

PubMed Central

Melendez, Johan H.; Santaus, Tonya M.; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A.; Geddes, Chris D.

2016-01-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving PCR-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (GC) DNA. Our approach is based on the use of highly-focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the present study, we show that highly focused microwaves at 2.45 GHz, using 12.3 mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification in less than 10 minutes total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

On the mechanism of Cr (VI)-induced carcinogenesis: dose dependence of uptake and cellular responses.

PubMed

Liu, K; Husler, J; Ye, J; Leonard, S S; Cutler, D; Chen, F; Wang, S; Zhang, Z; Ding, M; Wang, L; Shi, X

2001-06-01

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

PubMed Central

Fan, Wei; Wu, Xin; Ding, Baoyue; Gao, Jing; Cai, Zhen; Zhang, Wei; Yin, Dongfeng; Wang, Xiang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2012-01-01

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future. PMID:22403492

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model.

PubMed

Suarez, D L; Schultz-Cherry, S

2000-01-01

Vaccination of poultry with naked plasmid DNA has been successfully demonstrated with several different poultry pathogens, but the technology needs to be further developed before it can be practically implemented. Many different methods can conceivably enhance the efficacy of DNA vaccines, and this report examines the use of different eukaryotic expression vectors with different promoters and different adjuvants to express the influenza hemagglutinin protein. Four different promoters in five different plasmids were used to express the hemagglutinin protein of an H5 avian influenza virus, including two different immediate early cytomegaloviruses (CMVs), Rous sarcoma virus, chicken actin, and simian virus 40 promoters. All five constructs expressed detectable hemagglutinin protein in cell culture, but the pCI-neo HA plasmid with the CMV promoter provided the best response in chickens when vaccinated intramuscularly at 1 day of age on the basis of antibody titer and survivability after challenge with a highly pathogenic avian influenza virus at 6 wk postinoculation. A beneficial response was observed in birds boostered at 3 wk of age, in birds given larger amounts of DNA, and with the use of multiple injection sites to administer the vaccine. With the use of the pCI-neo construct, the effects of different adjuvants designed to increase the uptake of plasmid DNA, including 25% sucrose, diethylaminoethyl dextran, calcium phosphate, polybrene, and two different cationic liposomes, were examined. Both liposomes tested enhanced antibody titers as compared with the positive controls, but the other chemical adjuvants decreased the antibody response as compared with the control chickens that received just the plasmid alone. The results observed are promising for continued studies, but continued improvements in vaccine response and reduced costs are necessary before the technology can be commercially developed.

Assessment of environmental DNA for detecting presence of imperiled aquatic amphibian species in isolated wetlands

USGS Publications Warehouse

Mckee, Anna; Calhoun, Daniel L.; Barichivich, William J.; Spear, Stephen F.; Goldberg, Caren S.; Glenn, Travis C

2015-01-01

Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.

Development of Shuttle Vectors for Halobacteria

DTIC Science & Technology

1989-02-01

the time of application, we had completed a study of transfection, using Halobacterium halobiun and its phage ,DH. We showed (Cline and Doolittle... phage particles), producing plaques titratable on H. halobium lawns. Uptake of DNA by both species appears to be about equally efficient, but DNA from H...halobium-grown phage is restricted in H. volcanii, which shows a 104 - 105 reduction in transfection efficiency (400 plaques per pg) but no reduction

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

PubMed

Edelson, Benjamin S; Best, Timothy P; Olenyuk, Bogdan; Nickols, Nicholas G; Doss, Raymond M; Foister, Shane; Heckel, Alexander; Dervan, Peter B

2004-01-01

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

DNA Nanotechnology-Enabled Drug Delivery Systems.

PubMed

Hu, Qinqin; Li, Hua; Wang, Lihua; Gu, Hongzhou; Fan, Chunhai

2018-02-21

Over the past decade, we have seen rapid advances in applying nanotechnology in biomedical areas including bioimaging, biodetection, and drug delivery. As an emerging field, DNA nanotechnology offers simple yet powerful design techniques for self-assembly of nanostructures with unique advantages and high potential in enhancing drug targeting and reducing drug toxicity. Various sequence programming and optimization approaches have been developed to design DNA nanostructures with precisely engineered, controllable size, shape, surface chemistry, and function. Potent anticancer drug molecules, including Doxorubicin and CpG oligonucleotides, have been successfully loaded on DNA nanostructures to increase their cell uptake efficiency. These advances have implicated the bright future of DNA nanotechnology-enabled nanomedicine. In this review, we begin with the origin of DNA nanotechnology, followed by summarizing state-of-the-art strategies for the construction of DNA nanostructures and drug payloads delivered by DNA nanovehicles. Further, we discuss the cellular fates of DNA nanostructures as well as challenges and opportunities for DNA nanostructure-based drug delivery.

The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae.

PubMed

Blokesch, Melanie; Schoolnik, Gary K

2008-11-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.

The Extracellular Nuclease Dns and Its Role in Natural Transformation of Vibrio cholerae▿

PubMed Central

Blokesch, Melanie; Schoolnik, Gary K.

2008-01-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification. PMID:18757542

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

PubMed Central

Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

PubMed

Riva, Francesca; Bidard, Francois-Clement; Houy, Alexandre; Saliou, Adrien; Madic, Jordan; Rampanou, Aurore; Hego, Caroline; Milder, Maud; Cottu, Paul; Sablin, Marie-Paule; Vincent-Salomon, Anne; Lantz, Olivier; Stern, Marc-Henri; Proudhon, Charlotte; Pierga, Jean-Yves

2017-03-01

In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival. Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival. © 2016 American Association for Clinical Chemistry.

Understanding environmental DNA detection probabilities: A case study using a stream-dwelling char Salvelinus fontinalis

USGS Publications Warehouse

Wilcox, Taylor M; Mckelvey, Kevin S.; Young, Michael K.; Sepulveda, Adam; Shepard, Bradley B.; Jane, Stephen F; Whiteley, Andrew R.; Lowe, Winsor H.; Schwartz, Michael K.

2016-01-01

Environmental DNA sampling (eDNA) has emerged as a powerful tool for detecting aquatic animals. Previous research suggests that eDNA methods are substantially more sensitive than traditional sampling. However, the factors influencing eDNA detection and the resulting sampling costs are still not well understood. Here we use multiple experiments to derive independent estimates of eDNA production rates and downstream persistence from brook trout (Salvelinus fontinalis) in streams. We use these estimates to parameterize models comparing the false negative detection rates of eDNA sampling and traditional backpack electrofishing. We find that using the protocols in this study eDNA had reasonable detection probabilities at extremely low animal densities (e.g., probability of detection 0.18 at densities of one fish per stream kilometer) and very high detection probabilities at population-level densities (e.g., probability of detection > 0.99 at densities of ≥ 3 fish per 100 m). This is substantially more sensitive than traditional electrofishing for determining the presence of brook trout and may translate into important cost savings when animals are rare. Our findings are consistent with a growing body of literature showing that eDNA sampling is a powerful tool for the detection of aquatic species, particularly those that are rare and difficult to sample using traditional methods.

Field calibration of blowfly-derived DNA against traditional methods for assessing mammal diversity in tropical forests.

PubMed

Lee, Ping-Shin; Gan, Han Ming; Clements, Gopalasamy Reuben; Wilson, John-James

2016-11-01

Mammal diversity assessments based on DNA derived from invertebrates have been suggested as alternatives to assessments based on traditional methods; however, no study has field-tested both approaches simultaneously. In Peninsular Malaysia, we calibrated the performance of mammal DNA derived from blowflies (Diptera: Calliphoridae) against traditional methods used to detect species. We first compared five methods (cage trapping, mist netting, hair trapping, scat collection, and blowfly-derived DNA) in a forest reserve with no recent reports of megafauna. Blowfly-derived DNA and mist netting detected the joint highest number of species (n = 6). Only one species was detected by multiple methods. Compared to the other methods, blowfly-derived DNA detected both volant and non-volant species. In another forest reserve, rich in megafauna, we calibrated blowfly-derived DNA against camera traps. Blowfly-derived DNA detected more species (n = 11) than camera traps (n = 9), with only one species detected by both methods. The rarefaction curve indicated that blowfly-derived DNA would continue to detect more species with greater sampling effort. With further calibration, blowfly-derived DNA may join the list of traditional field methods. Areas for further investigation include blowfly feeding and dispersal biology, primer biases, and the assembly of a comprehensive and taxonomically-consistent DNA barcode reference library.

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

USGS Publications Warehouse

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Enzyme-free detection and quantification of double-stranded nucleic acids.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

2012-08-01

We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

PubMed

de Souza, Lesley S; Godwin, James C; Renshaw, Mark A; Larson, Eric

2016-01-01

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms

PubMed Central

de Souza, Lesley S.; Godwin, James C.; Renshaw, Mark A.; Larson, Eric

2016-01-01

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs. PMID:27776150

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

PubMed

Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

2016-10-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

PubMed

Pezo, Valérie; Hassan, Camille; Louis, Dominique; Sargueil, Bruno; Herdewijn, Piet; Marlière, Philippe

2018-05-18

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

[11C]choline uptake in regenerating liver after partial hepatectomy or CCl4-administration.

PubMed

Sasaki, Toru

2004-02-01

To characterize [methyl-(11)C]choline ([(11)C]choline) as an oncologic PET radiopharmaceutical, [(11)C]choline uptake in regenerating livers after partial hepatectomy as a model of typical proliferating tissue and after CCl(4) insult as that of proliferating tissue with inflammation, was studied in rats. [(11)C]Choline, [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) and [2-(14)C]thymidine ([(14)C]TdR) uptake was studied in regenerating rat liver after 70% partial hepatectomy or CCl(4)-administration. [(11)C]Choline uptake in regenerating liver after partial hepatectomy was significantly increased with [(14)C]TdR uptake as a marker of DNA synthesis at 18 hours after surgery. On the other hand, the uptake was not accelerated by CCl(4)-administration, though it significantly increased [(14)C]TdR uptake. There were no differences of [(11)C]choline uptake acceleration following partial hepatectomy among the three parts of the regenerating liver. [(18)F]FDG uptake was accelerated in the regenerating liver on either partial hepatectomy or CCl(4)-administration. The magnitude of the increase in [(18)F]FDG uptake in the regenerating liver induced by partial hepatectomy was greater than that for [(11)C]choline. [(11)C]Choline uptake in the liver was accelerated by partial hepatectomy, but not by CCl(4)-administration. This might be expected given that the differentiation between proliferating tissues such as tumor and inflammatory tissue was possible by [(11)C]choline-PET.

Cyclen-based lipidic oligomers as potential gene delivery vehicles.

PubMed

Yi, Wen-Jing; Zhang, Qin-Fang; Zhang, Ji; Liu, Qiang; Ren, Laifeng; Chen, Qian-Ming; Guo, Liandi; Yu, Xiao-Qi

2014-03-01

A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

PubMed Central

Luo, Jun; Cui, Xiuji; Gao, Lu

2017-01-01

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3′ exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3′ end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5′ end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC DNA to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. PMID:28637752

The detection of (total and ccc) HBV DNA in liver transplant recipients with hepatitis B vaccine against HBV reinfection.

PubMed

Duan, Bin-Wei; Lu, Shi-Chun; Lai, Wei; Liu, Xue-En; Liu, Yuan

2015-01-01

To investigate the levels of hepatitis B virus total DNA (HBV DNA) and covalently closed circular (ccc) DNA in liver transplant recipients who received hepatitis B vaccination, including responders and non-responders, following liver transplantation due to hepatitis B-related diseases and to investigate the efficacy of hepatitis B immune reconstitution against HBV reinfection. Twenty responders and 34 non-responders were enrolled in the present study. The levels of HBV total DNA and ccc DNA in peripheral blood mononuclear cells (PBMCs) and the liver and plasma were detected by real-time polymerase chain reaction (PCR). Fifty-three blood samples and 38 liver allograft tissues were acquired. For the responders, the mean serum titer for anti-HBs (antibodies against hepatitis B surface antigen) was 289 (46.64-1000) IU/ml. Also for the responders, HBV total DNA was detected in PBMCs for one recipient and in the liver for another recipient, but ccc DNA was not detected in either of those 2 recipients. For the non-responders, HBV total DNA was detected in PBMCS for 2 recipients, neither of whom had ccc DNA. Also for the non-responders, HBV total DNA was detected in the livers of 3 recipients, 2 of whom also had ccc DNA. All responders had discontinued hepatitis B immunoglobulin (HBIG), and 13 responders had discontinued antiviral agents. One responder experienced HBV recurrence during the follow-up period. For the majority of liver transplant recipients, no HBV total DNA or ccc DNA was detected in the blood or liver. The lack of HBV total DNA and ccc DNA both in PBMCs and the liver in liver transplant recipients who received hepatitis B vaccination to prevent HBV reinfection should be a prerequisite for the withdrawal of HBIG and/or antiviral agents.

Tazarotene-Induced Gene 1 Interacts with DNAJC8 and Regulates Glycolysis in Cervical Cancer Cells.

PubMed

Wang, Chun-Hua; Shyu, Rong-Yaun; Wu, Chang-Chieh; Chen, Mao-Liang; Lee, Ming-Cheng; Lin, Yi-Yin; Wang, Lu-Kai; Jiang, Shun-Yuan; Tsai, Fu-Ming

2018-06-14

The tazarotene-induced gene 1 (TIG1) protein is a retinoidinducible growth regulator and is considered a tumor suppressor. Here, we show that DnaJ heat shock protein family member C8 (DNAJC8) is a TIG1 target that regulates glycolysis. Ectopic DNAJC8 expression induced the translocation of pyruvate kinase M2 (PKM2) into the nucleus, subsequently inducing glucose transporter 1 (GLUT1) expression to promote glucose uptake. Silencing either DNAJC8 or PKM2 alleviated the upregulation of GLUT1 expression and glucose uptake induced by ectopic DNAJC8 expression. TIG1 interacted with DNAJC8 in the cytosol, and this interaction completely blocked DNAJC8-mediated PKM2 translocation and inhibited glucose uptake. Furthermore, increased glycose uptake was observed in cells in which TIG1 was silenced. In conclusion, TIG1 acts as a pivotal repressor of DNAJC8 to enhance glucose uptake by partially regulating PKM2 translocation.

Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

PubMed

Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

2015-02-15

A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA.

PubMed

Mao, Pingdao; Ning, Yi; Li, Wenkai; Peng, Zhihui; Chen, Yongzhe; Deng, Le

2014-01-10

A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of DNA methylation profile on OATP3A1 and OATP4A1 transcript levels in colorectal cancer.

PubMed

Rawłuszko-Wieczorek, Agnieszka Anna; Horst, Nikodem; Horbacka, Karolina; Bandura, Artur Szymon; Świderska, Monika; Krokowicz, Piotr; Jagodziński, Paweł Piotr

2015-08-01

Epidemiological studies indicate that 17β-estradiol (E2) prevents colorectal cancer (CRC). Organic anion transporting polypeptides (OATPs) are involved in the cellular uptake of various endogenous and exogenous substrates, including hormone conjugates. Because transfer of estrone sulfate (E1-S) can contribute to intra-tissue conversion of estrone to the biologically active form -E2, it is evident that the expression patterns of OATPs may be relevant to the analysis of CRC incidence and therapy. We therefore evaluated DNA methylation and transcript levels of two members of the OATP family, OATP3A1 and OATP4A1, that may be involved in E1-S transport in colorectal cancer patients. We detected a significant reduction in OATP3A1 and a significant increase in OATP4A1 mRNA levels in cancerous tissue, compared with histopathologically unchanged tissue (n=103). Moreover, we observed DNA hypermethylation in the OATP3A1 promoter region in a small subset of CRC patients and in HCT116 and Caco-2 colorectal cancer cell lines. We also observed increased OATP3A1 transcript following treatment with 5-aza-2-deoxycytidine and sodium butyrate. The OATP4A1 promoter region was hypomethylated in analyzed tissues and CRC cell lines and was not affected by these treatments. Our results suggest a potential mechanism for OATP3A1 downregulation that involves DNA methylation during colorectal carcinogenesis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

PubMed

Bakhori, Noremylia Mohd; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-12-12

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10-9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

PubMed

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-12-01

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

Assessment of circulating copy number variant detection for cancer screening.

PubMed

Molparia, Bhuvan; Nichani, Eshaan; Torkamani, Ali

2017-01-01

Current high-sensitivity cancer screening methods, largely utilizing correlative biomarkers, suffer from false positive rates that lead to unnecessary medical procedures and debatable public health benefit overall. Detection of circulating tumor DNA (ctDNA), a causal biomarker, has the potential to revolutionize cancer screening. Thus far, the majority of ctDNA studies have focused on detection of tumor-specific point mutations after cancer diagnosis for the purpose of post-treatment surveillance. However, ctDNA point mutation detection methods developed to date likely lack either the scope or analytical sensitivity necessary to be useful for cancer screening, due to the low (<1%) ctDNA fraction derived from early stage tumors. On the other hand, tumor-derived copy number variant (CNV) detection is hypothetically a superior means of ctDNA-based cancer screening for many tumor types, given that, relative to point mutations, each individual tumor CNV contributes a much larger number of ctDNA fragments to the overall pool of circulating free DNA (cfDNA). A small number of studies have demonstrated the potential of ctDNA CNV-based screening in select cancer types. Here we perform an in silico assessment of the potential for ctDNA CNV-based cancer screening across many common cancers, and suggest ctDNA CNV detection shows promise as a broad cancer screening methodology.

Lanthanoplatins: emissive Eu(iii) and Tb(iii) complexes staining nucleoli targeted through Pt-DNA crosslinking.

PubMed

Singh, Khushbu; Singh, Swati; Srivastava, Payal; Sivakumar, Sri; Patra, Ashis K

2017-06-01

Two highly luminescent water-soluble heterometallic LnPt 2 complexes, [{cis-PtCl(NH 3 ) 2 } 2 Ln(L)(H 2 O)](NO 3 ) 2 (Ln = Eu (1), Tb (2)), have been designed for their selective nucleoli staining through formation of Pt-DNA crosslinks. The complexes showed significant cellular uptake and distinctive nucleoli localization through intrinsic emission from Eu III or Tb III observed through confocal fluorescence microscopy.

Comparing efficiency of American Fisheries Society standard snorkeling techniques to environmental DNA sampling techniques

USGS Publications Warehouse

Ulibarri, Roy M.; Bonar, Scott A.; Rees, Christopher B.; Amberg, Jon J.; Ladell, Bridget; Jackson, Craig

2017-01-01

Analysis of environmental DNA (eDNA) is an emerging technique used to detect aquatic species through water sampling and the extraction of biological material for amplification. Our study compared the efficacy of eDNA methodology to American Fisheries Society (AFS) standard snorkeling surveys with regard to detecting the presence of rare fish species. Knowing which method is more efficient at detecting target species will help managers to determine the best way to sample when both traditional sampling methods and eDNA sampling are available. Our study site included three Navajo Nation streams that contained Navajo Nation Genetic Subunit Bluehead Suckers Catostomus discobolus and Zuni Bluehead Suckers C. discobolus yarrowi. We first divided the entire wetted area of streams into consecutive 100-m reaches and then systematically selected 10 reaches/stream for snorkel and eDNA surveys. Surface water samples were taken in 10-m sections within each 100-m reach, while fish presence was noted via snorkeling in each 10-m section. Quantitative PCR was run on each individual water sample in quadruplicate to test for the presence or absence of the target species. With eDNA sampling techniques, we were able to positively detect both species in two out of the three streams. Snorkeling resulted in positive detection of both species in all three streams. In streams where the target species were detected with eDNA sampling, snorkeling detected fish at 11–29 sites/stream, whereas eDNA detected fish at 3–12 sites/stream. Our results suggest that AFS standard snorkeling is more effective than eDNA for detecting target fish species. To improve our eDNA procedures, the amount of water collected and tested should be increased. Additionally, filtering water on-site may improve eDNA techniques for detecting fish. Future research should focus on standardization of eDNA sampling to provide a widely operational sampling tool.

Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

PubMed

Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

2015-05-05

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

Digital DNA detection based on a compact optofluidic laser with ultra-low sample consumption.

PubMed

Lee, Wonsuk; Chen, Qiushu; Fan, Xudong; Yoon, Dong Ki

2016-11-29

DNA lasers self-amplify optical signals from a DNA analyte as well as thermodynamic differences between sequences, allowing quasi-digital DNA detection. However, these systems have drawbacks, such as relatively large sample consumption and complicated dye labelling. Moreover, although the lasing signal can detect the target DNA, it is superimposed on an unintended fluorescence background, which persists for non-target DNA samples as well. From an optical point of view, it is thus not truly digital detection and requires spectral analysis to identify the target. In this work, we propose and demonstrate an optofluidic laser that has a single layer of DNA molecules as the gain material. A target DNA produces intensive laser emission comparable to existing DNA lasers, while any unnecessary fluorescence background is successfully suppressed. As a result, the target DNA can be detected with a single laser pulse, in a truly digital manner. Since the DNA molecules cover only a single layer on the surface of the laser microcavity, the DNA sample consumption is a few orders of magnitude lower than that of existing DNA lasers. Furthermore, the DNA molecules are stained by simply immersing the microcavity in the intercalating dye solution, and thus the proposed DNA laser is free of any complex dye-labelling process prior to analysis.

Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

PubMed Central

Liu, Bing; Li, Lei; Huang, Lixia; Li, Shaoli; Rao, Guanhua; Yu, Yang; Zhou, Yanbin

2017-01-01

Emerging evidence has indicated that circulating tumor DNA (ctDNA) from plasma could be used to analyze EGFR mutation status for NSCLC patients; however, due to the low level of ctDNA in plasma, highly sensitive approaches are required to detect low frequency mutations. In addition, the cutoff for the mutation abundance that can be detected in tumor tissue but cannot be detected in matched ctDNA is still unknown. To assess a highly sensitive method, we evaluated the use of digital PCR in the detection of EGFR mutations in tumor tissue from 47 advanced lung adenocarcinoma patients through comparison with NGS and ARMS. We determined the degree of concordance between tumor tissue DNA and paired ctDNA and analyzed the mutation abundance relationship between them. Digital PCR and Proton had a high sensitivity (96.00% vs. 100%) compared with that of ARMS in the detection of mutations in tumor tissue. Digital PCR outperformed Proton in identifying more low abundance mutations. The ctDNA detection rate of digital PCR was 87.50% in paired tumor tissue with a mutation abundance above 5% and 7.59% in paired tumor tissue with a mutation abundance below 5%. When the DNA mutation abundance of tumor tissue was above 3.81%, it could identify mutations in paired ctDNA with a high sensitivity. Digital PCR will help identify alternative methods for detecting low abundance mutations in tumor tissue DNA and plasma ctDNA. PMID:28978074

Cerebral utilization of glucose, ketone bodies and oxygen in starving infant rats and the effect of intrauterine growth retardation.

PubMed

Dahlquist, G

1976-10-01

Cerebral arteriovenous differences of acetoacetate, D-beta-hydroxybutyrate, glucose, lactate and oxygen and brain DNA content was measured at 20 days of age in intrauterine growth retarded (IUGR) rats and normal littermates after 48 and 72 h of starvation. Cerebral blood flow (CBF) was measured with labeled microspheres in other comparable groups of IUGR and control rats. CBF was similar in IUGR and normal littermates (0.57+/-0.09 and 0.58+/-0.10 ml/min respectively). After 48 h of starvation, arterial glucose was significantly lower in IUGR than control animals but the arterial concentrations of ketone bodies were similar. After 48 h of starvation, cerebral arteriovenous difference of beta-hydroxybutyrate was significantly higher in control than IUGR rats also when expressed per mg brain DNA as was the fractional uptake of D-beta-hydroxybutyrate. After 72 h of starvation, arterial concentrations of ketone bodies were significantly lower in IUGR rats than controls but the fractional uptake of D-beta-hydroxybutyrate was increased compared to IUGR rats starved for 48 h. The average percentage of calculated total substrate uptake (mumol/min) accounted for by ketone bodies increased in control animals from 31.1% after 48 h of starvation to 41.0% after 72 h of starvation. In IUGR rats these percentage values were 26.5 and 25.7 respectively. After 72 h of starvation the fraction of total cerebral uptake of substrates accounted for by ketone bodies was significantly higher in control that IUGR rats. As total cerebral uptake of substrates was similar between IUGR and control animals it is concluded that IUGR rats are more dependent on glucose as a substrate for the brain during starvation.

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

PubMed

Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

2017-06-21

The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. Copyright © 2017 American Society for Microbiology.

Evidence of the differential biotransformation and genotoxicity of ZnO and CeO2 nanoparticles on soybean (Glycine max) plants.

PubMed

López-Moreno, Martha L; de la Rosa, Guadalupe; Hernández-Viezcas, José A; Castillo-Michel, Hiram; Botez, Cristian E; Peralta-Videa, José R; Gardea-Torresdey, Jorge L

2010-10-01

Concern and interest related to the effects of nanomaterials on living organisms are growing in both the scientific and public communities. Reports have described the toxicity of nanoparticles (NPs) on micro- and macro-organisms, including some plant species. Nevertheless, to the authors' knowledge there are no reports on the biotransformation of NPs by edible terrestrial plants. Here, shown for the first time, is evidence pertaining to the biotransformation of ZnO and CeO(2) NPs in plant seedlings. Although the NPs did not affect soybean germination, they produced a differential effect on plant growth and element uptake. By using synchrotron X-ray absorption spectroscopy we obtained clear evidence of the presence of CeO(2) NPs in roots, whereas ZnO NPs were not present. Random amplified polymorphic DNA assay was applied to detect DNA damage and mutations caused by NPs. Results obtained from the exposure of soybean plants to CeO(2) NPs show the appearance of four new bands at 2000 mg L(-1) and three new bands at 4000 mg L(-1) treatment. In this study we demonstrated genotoxic effects from the exposure of soybean plants to CeO(2) NPs.

Long term effects of neonatal hypoglycaemia on pancreatic function.

PubMed

Anju, T R; Paulose, C S

2015-02-01

Low blood glucose in neonates predisposes to long term pancreatic damage. We focused on evaluating long term consequences of neonatal hypoglycaemia in pancreatic functions. Pancreatic function was analysed by measuring DNA/protein synthesis, glucose/ATP uptake in vitro. Gene expression of Pdx1, NeuroD1, Pax4, Bax, caspase 3, Beclin1 were done. Muscarinic receptors were analysed by radio receptor assay. Overall pancreatic efficiency was reduced in one-month-old rats exposed to neonatal hypoglycaemia as indicated by decreased DNA/protein synthesis and glucose/ATP uptake in vitro. Both Pdx1 and Neuro D1 expression were significantly down-regulated whereas Pax4 was up-regulated. Up-regulated Bax, caspase 3 and beclin1 along with reduced muscarinic receptors accounts for activation of cell death pathways. The study revealed a drastic reduction in pancreatic functions along with activation of apoptotic factors in one month old rats exposed to neonatal hypoglycaemia.

Mutation-based detection and monitoring of cell-free tumor DNA in peripheral blood of cancer patients.

PubMed

Benesova, L; Belsanova, B; Suchanek, S; Kopeckova, M; Minarikova, P; Lipska, L; Levy, M; Visokai, V; Zavoral, M; Minarik, M

2013-02-15

Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future. Copyright © 2012 Elsevier Inc. All rights reserved.

Environmental DNA (eDNA) detects the invasive rusty crayfish Orconectes rusticus at low abundances.

PubMed

Dougherty, Matthew M; Larson, Eric R; Renshaw, Mark A; Gantz, Crysta A; Egan, Scott P; Erickson, Daniel M; Lodge, David M

2016-06-01

Early detection is invaluable for the cost-effective control and eradication of invasive species, yet many traditional sampling techniques are ineffective at the low population abundances found at the onset of the invasion process. Environmental DNA (eDNA) is a promising and sensitive tool for early detection of some invasive species, but its efficacy has not yet been evaluated for many taxonomic groups and habitat types.We evaluated the ability of eDNA to detect the invasive rusty crayfish Orconectes rusticus and to reflect patterns of its relative abundance, in upper Midwest, USA, inland lakes. We paired conventional baited trapping as a measure of crayfish relative abundance with water samples for eDNA, which were analysed in the laboratory with a qPCR assay. We modelled detection probability for O. rusticus eDNA using relative abundance and site characteristics as covariates and also tested the relationship between eDNA copy number and O. rusticus relative abundance.We detected O. rusticus eDNA in all lakes where this species was collected by trapping, down to low relative abundances, as well as in two lakes where trap catch was zero. Detection probability of O. rusticus eDNA was well predicted by relative abundance of this species and lake water clarity. However, there was poor correspondence between eDNA copy number and O. rusticus relative abundance estimated by trap catches. Synthesis and applications . Our study demonstrates a field and laboratory protocol for eDNA monitoring of crayfish invasions, with results of statistical models that provide guidance of sampling effort and detection probabilities for researchers in other regions and systems. We propose eDNA be included as a tool in surveillance for invasive or imperilled crayfishes and other benthic arthropods.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Persistence of environmental DNA in freshwater ecosystems.

PubMed

Dejean, Tony; Valentini, Alice; Duparc, Antoine; Pellier-Cuit, Stéphanie; Pompanon, François; Taberlet, Pierre; Miaud, Claude

2011-01-01

The precise knowledge of species distribution is a key step in conservation biology. However, species detection can be extremely difficult in many environments, specific life stages and in populations at very low density. The aim of this study was to improve the knowledge on DNA persistence in water in order to confirm the presence of the focus species in freshwater ecosystems. Aquatic vertebrates (fish: Siberian sturgeon and amphibian: Bullfrog tadpoles) were used as target species. In control conditions (tanks) and in the field (ponds), the DNA detectability decreases with time after the removal of the species source of DNA. DNA was detectable for less than one month in both conditions. The density of individuals also influences the dynamics of DNA detectability in water samples. The dynamics of detectability reflects the persistence of DNA fragments in freshwater ecosystems. The short time persistence of detectable amounts of DNA opens perspectives in conservation biology, by allowing access to the presence or absence of species e.g. rare, secretive, potentially invasive, or at low density. This knowledge of DNA persistence will greatly influence planning of biodiversity inventories and biosecurity surveys.

Detection of DNA hybridization using graphene-coated black phosphorus surface plasmon resonance sensor

NASA Astrophysics Data System (ADS)

Pal, Sarika; Verma, Alka; Raikwar, S.; Prajapati, Y. K.; Saini, J. P.

2018-05-01

In this paper, graphene-coated black phosphorus at the metal surface for the detection of DNA hybridization event is numerically demonstrated. The strategy consists of placing the sensing medium on top of black phosphorus-graphene-coated SPR which interfaces with phosphate-buffered saline solution carrying single-stranded DNA. Upon hybridization with its complementary DNA, desorption of the nanostructures takes place and thus enables the sensitive detection of the DNA hybridization event. The proposed sensor exhibits a sensitivity (125 ο/RIU), detection accuracy (0.95) and quality factor (13.62 RIU-1) for complementary DNA. In comparison with other reported papers, our suggested sensor provides much better performance. Thus, this label-free DNA detection platform should spur off new interest towards the use of black phosphorus-graphene-coated SPR interfaces.

[Real-time PCR kits for the detection of the African Swine Fever virus].

PubMed

Latyshev, O E; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Tsibezov, V V; Chernykh, O Iu; Dzhailidi, G A; Aliper, T I

2014-01-01

The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.

Circulating androgen receptor combined with 18F-fluorocholine PET/CT metabolic activity and outcome to androgen receptor signalling-directed therapies in castration-resistant prostate cancer.

PubMed

Conteduca, V; Scarpi, E; Caroli, P; Salvi, S; Lolli, C; Burgio, S L; Menna, C; Schepisi, G; Testoni, S; Gurioli, G; Paganelli, G; Casadio, V; Matteucci, F; De Giorgi, U

2017-11-14

The association between choline uptake and androgen receptor (AR) expression is suggested by the upregulation of choline kinase-alpha in prostate cancer. Recently, detection of AR aberration in cell-free DNA as well as early 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) were associated with outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone and enzalutamide. We aimed to make a direct comparison between circulating AR copy number (CN) and choline uptake at FCH-PET/CT. We analysed 80 mCRPC patients progressing after docetaxel treated with abiraterone (n = 47) or enzalutamide (n = 33). We analysed AR CN from plasma samples using digital PCR and Taqman CN assays and total lesion activity (TLA) and metabolic tumor volume (MTV) on FCH-PET/CT at baseline. A meaningful correlation was showed among AR gain and TLA/MTV compared to AR non-gained cases (P = 0.001 and P = 0.004, respectively), independently from type of treatment. Multivariate analysis revealed that AR CN and only TLA were associated with both shorter PFS (P < 0.0009 and P = 0.026, respectively) and OS (P < 0.031 and P = 0.039, respectively). AR gain appeared significantly correlated with choline uptake represented mainly by TLA. Further prospective studies are warranted to better address this pathway of AR-signalling and to identify multiplex biomarker strategies including plasma AR and FCH-PET/CT in mCRPC patients.

Understanding environmental DNA detection probabilities: A case study using a stream-dwelling char Salvelinus fontinalis

Treesearch

Taylor M. Wilcox; Kevin S. McKelvey; Michael K. Young; Adam J. Sepulveda; Bradley B. Shepard; Stephen F. Jane; Andrew R. Whiteley; Winsor H. Lowe; Michael K. Schwartz

2016-01-01

Environmental DNA sampling (eDNA) has emerged as a powerful tool for detecting aquatic animals. Previous research suggests that eDNA methods are substantially more sensitive than traditional sampling. However, the factors influencing eDNA detection and the resulting sampling costs are still not well understood. Here we use multiple experiments to derive...

Detection of parvovirus B19 DNA in blood: Viruses or DNA remnants?

PubMed

Molenaar-de Backer, M W A; Russcher, A; Kroes, A C M; Koppelman, M H G M; Lanfermeijer, M; Zaaijer, H L

2016-11-01

Parvovirus B19 (B19V) DNA can be detected in blood over a long period after acute infection. Several reports associate the presence of B19V DNA with disease, irrespective of timing of the initial B19V infection. This study aims to analyze the properties of B19V DNA in blood, differentiating between bare, non-infectious strands of DNA and B19V DNA in viable virions. Ten blood donors with asymptomatic acute B19V infection were followed and sampled up to 22 months after infection. The samples were treated with and without an endonuclease and tested for B19V DNA, to distinguish between DNA in virions and naked DNA. In the acute phase of infection, high levels of B19V DNA were detected, concurrent with B19V IgM antibodies. B19V DNA apparently was encapsidated, as indicated by resistance to endonuclease degradation. Subsequently, B19V DNA remained detectable for more than one year in all donors at low levels (<10 5 IU/mL). Approximately 150days after infection B19V DNA became degradable by an endonuclease, indicating that this concerned naked DNA. In some donors a second endonuclease-resistant peak occurred. Detection of B19V DNA in blood by PCR does not necessarily imply that B19V replication takes place and that infectious B19V virions are present. We propose that remnant B19V DNA strands can be released from tissues without active replication. This finding urges to reconsider an assumed role of B19V infection mainly based on B19V DNA detection in blood, a much debated subject in clinical syndromes such as myocarditis and arthritis. Copyright © 2016 Elsevier B.V. All rights reserved.

Residual eDNA detection sensitivity assessed by quantitative real-time PCR in a river ecosystem.

PubMed

Balasingham, Katherine D; Walter, Ryan P; Heath, Daniel D

2017-05-01

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real-time polymerase chain reaction (qRT-PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over 2 years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 h after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 h after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT-PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species. © 2016 John Wiley & Sons Ltd.

Detection of human papillomavirus DNA in urine. A review of the literature.

PubMed

Vorsters, A; Micalessi, I; Bilcke, J; Ieven, M; Bogers, J; Van Damme, P

2012-05-01

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

How to limit false positives in environmental DNA and metabarcoding?

PubMed

Ficetola, Gentile Francesco; Taberlet, Pierre; Coissac, Eric

2016-05-01

Environmental DNA (eDNA) and metabarcoding are boosting our ability to acquire data on species distribution in a variety of ecosystems. Nevertheless, as most of sampling approaches, eDNA is not perfect. It can fail to detect species that are actually present, and even false positives are possible: a species may be apparently detected in areas where it is actually absent. Controlling false positives remains a main challenge for eDNA analyses: in this issue of Molecular Ecology Resources, Lahoz-Monfort et al. () test the performance of multiple statistical modelling approaches to estimate the rate of detection and false positives from eDNA data. Here, we discuss the importance of controlling for false detection from early steps of eDNA analyses (laboratory, bioinformatics), to improve the quality of results and allow an efficient use of the site occupancy-detection modelling (SODM) framework for limiting false presences in eDNA analysis. © 2016 John Wiley & Sons Ltd.

Multicolour probes for sequence-specific DNA detection based on graphene oxide.

PubMed

Zhu, Qing; Xiang, Dongshan; Zhang, Cuiling; Ji, Xinghu; He, Zhike

2013-09-21

The bifunctionality of graphene oxide (GO) which can highly adsorb single-stranded DNA (ssDNA) and effectively quench the emission of organic dyes is reasonably utilized in a multiplexed DNA detection system, achieving sensitive and selective detection of HIV, VV and EV, respectively.

Renal amyloidosis. Evaluation by gallium imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, V.W.; Skinner, M.; Cohen, A.S.

1986-09-01

A study has been performed to evaluate the efficacy of gallium imaging in the detection of renal amyloidosis. Ten of the 11 patients who had biopsy-proven renal amyloidosis demonstrated marked uptake in both kidneys. One patient revealed moderate gallium uptake in his kidneys. None of the patients had underlying renal or extrarenal pathology other than amyloidosis, which could account for renal gallium uptake (renal infection, neoplasm, hepatic failure or frequent blood transfusions). Four patients also had extrarenal foci of abnormal gallium uptake, suggesting other sites of amyloid deposits. Our data strongly suggest that gallium imaging has a high sensitivity formore » detection of renal amyloidosis. Its specificity is enhanced significantly by careful review of the clinical history to exclude other known causes of renal gallium uptake. Potentially, gallium imaging may be used to monitor the progress of patients under experimental therapy.« less

Environmental DNA method for estimating salamander distribution in headwater streams, and a comparison of water sampling methods.

PubMed

Katano, Izumi; Harada, Ken; Doi, Hideyuki; Souma, Rio; Minamoto, Toshifumi

2017-01-01

Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water.

Simultaneous quantitative determination of 5-aza-2′-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

PubMed Central

Anders, Nicole M.; Liu, Jianyong; Wanjiku, Teresia; Giovinazzo, Hugh; Zhou, Jianya; Vaghasia, Ajay; Nelson, William G.; Yegnasubramanian, Srinivasan; Rudek, Michelle A.

2016-01-01

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2′-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2′-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2′-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100 mm × 2.1 mm, 5μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 minute total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2′-deoxycytidine, 2′-deoxycytidine, and 5-methyl-2′-deoxycytidine. The assay range was 2 – 400 ng/mL for 5-aza-2′-deoxycytidine, 50 – 10,000 ng/mL for 2′-deoxycytidine, and was 5 – 1,000 ng/mL for 5-methyl-2′-deoxycytidine. The assay proved to be accurate (93.0–102.2%) and precise (CV ≤ 6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at −70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2′-deoxycytidine and 5-methyl-2′-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine. PMID:27082761

Alteration of Membrane Permeability of Bacteria and Yeast by High Frequency Alternating Current (HFAC)

PubMed Central

Cassanelli, C; Marchese, A; Cagnacci, S; Debbia, E.A

2008-01-01

Aims: Endox® Endodontic System (Endox) is used for endodontic treatment by a high frequency alternating current (HFAC). This device damaged the envelopes of spores and vegetative organisms. If the integrity of the envelope is compromised, the transit of compounds in the two directions is possible. This latter aspect was investigated here. Methods: The instrument delivered a 60ms pulse at a frequency 300 kHz, and power 800 KV/m. DNA transfer was verified using Escherichia coli K-12 strain carrying a non conjugative plasmid pBP517 (gyrA+) as donor and a rifampicin and nalidixic acid resistant recipient. 0.2 ml of mixture of donor and recipient strains in saline was exposed to HFAC and plated on selective media. Uptake of antimicrobials and a delay in re-growth was assessed exposing the strains to HFAC. Results: Plasmid transfer was detected under different experimental conditions. From 9 to 27 recombinants were found. Representative recombinants cured from plasmid showed the original phenotype. HFAC promoted the uptake of ineffective antibiotics, and induces a 1 h of delay in re-growth on the strains. Conclusions: Endox exhibited an effect on microrganisms which is reminiscent with that occuring in electroporation, but with a mode of action that saved materials and time. PMID:19088909

Characterization of a Vibrio vulnificus LysR homologue, HupR, which regulates expression of the haem uptake outer membrane protein, HupA.

PubMed

Litwin, C M; Quackenbush, J

2001-12-01

In Vibrio vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. Here, we show that the DNA upstream of hupA (haem uptake receptor) in V. vulnificus encodes a protein in the inverse orientation to hupA (named hupR). HupR shares homology with the LysR family of positive transcriptional activators. A hupA-lacZ fusion contained on a plasmid was transformed into Fur(-), Fur(+)and HupR(-)strains of V. vulnificus. The beta-galactosidase assays and Northern blot analysis showed that transcription of hupA is negatively regulated by iron and the Fur repressor in V. vulnificus. Under low-iron conditions with added haemin, the expression of hupA in the hupR mutant was significantly lower than in the wild-type. This diminished response to haem was detected by both Northern blot and hupA-lacZ fusion analysis. The haem response of hupA in the hupR mutant was restored to wild-type levels when complemented with hupR in trans. These studies suggest that HupR may act as a positive regulator of hupA transcription under low-iron conditions in the presence of haemin. Copyright 2001 Academic Press.

C. elegans-on-a-chip for in situ and in vivo Ag nanoparticles’ uptake and toxicity assay

NASA Astrophysics Data System (ADS)

Kim, Jin Ho; Lee, Seung Hwan; Cha, Yun Jeong; Hong, Sung Jin; Chung, Sang Kug; Park, Tai Hyun; Choi, Shin Sik

2017-01-01

Nanomaterials are extensively used in consumer products and medical applications, but little is known about their environmental and biological toxicities. Moreover, the toxicity analysis requires sophisticated instruments and labor-intensive experiments. Here we report a microfluidic chip incorporated with the nematode Caenorhabditis elegans that rapidly displays the changes in body growth and gene expression specifically responsive to the silver nanoparticles (AgNPs). C. elegans were cultured in microfluidic chambers in the presence or absence of AgNPs and were consequently transferred to wedge-shaped channels, which immobilized the animals, allowing the evaluation of parameters such as length, moving distance, and fluorescence from the reporter gene. The AgNPs reduced the length of C. elegans body, which was easily identified in the channel of chip. In addition, the decrease of body width enabled the worm to advance the longer distance compared to the animal without nanoparticles in a wedge-shaped channel. The transgenic marker DNA, mtl-2::gfp was highly expressed upon the uptake of AgNPs, resulting in green fluorescence emission. The comparative investigation using gold nanoparticles and heavy-metal ions indicated that these parameters are specific to AgNPs. These results demonstrate that C. elegans-on-a-chip has a great potential as a rapid and specific nanoparticle detection or nanotoxicity assessment system.

Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubio, Laura; El Yamani, Naouale; Kazimirova, Alena

Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12 µg/cm{sup 2} At these rangemore » we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure. - Highlights: • MWCNT were tested in V79 cells. • Cellular uptake of MWCNT was detected using TEM. • Intracellular ROS induction was observed after MWCNT exposure. • MWCNT induced a concentration-dependent increase of HPRT mutations.« less

The utilization of circulating cell-free fetal DNA testing and decrease in invasive diagnostic procedures: an institutional experience.

PubMed

Pettit, K E; Hull, A D; Korty, L; Jones, M C; Pretorius, D H

2014-10-01

To characterize the patient population utilizing circulating cell-free fetal DNA (ccffDNA) testing at a large academic center and evaluate trends in the performance of invasive diagnostic procedures. A retrospective cohort study of all patients who underwent cell-free DNA testing from May to December 2012 was performed. During the study period, 206 patients had cell-free DNA testing. Of those, 75% (155/206) were of ages ⩾ 35 years. Of those undergoing ccffDNA testing, 41% had positive aneuploidy screening and 38% had abnormal ultrasound findings. Only 7% of the patients with negative ccffDNA testing opted for an invasive diagnostic procedure compared with 60% with positive testing (P<0.01). The rate of invasive procedures decreased from 5.9% of all visits to the center during a similar 8-month period in 2010 to 4.1% of all visits during the study period (P<0.01). Our data suggest that ccffDNA testing leads to reduced uptake of invasive procedures.

Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA.

PubMed

Piaggio, Antoinette J; Engeman, Richard M; Hopken, Matthew W; Humphrey, John S; Keacher, Kandy L; Bruce, William E; Avery, Michael L

2014-03-01

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Development of 19F-NMR chemical shift detection of DNA B-Z equilibrium using 19F-NMR.

PubMed

Nakamura, S; Yang, H; Hirata, C; Kersaudy, F; Fujimoto, K

2017-06-28

Various DNA conformational changes are in correlation with biological events. In particular, DNA B-Z equilibrium showed a high correlation with translation and transcription. In this study, we developed a DNA probe containing 5-trifluoromethylcytidine or 5-trifluoromethylthymidine to detect DNA B-Z equilibrium using 19 F-NMR. Its probe enabled the quantitative detection of B-, Z-, and ss-DNA based on 19 F-NMR chemical shift change.

Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

PubMed Central

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

2013-01-01

An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense. PMID:25587406

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

PubMed

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

UPTAKE OF BACTERIOPHAGE F2 THROUGH PLANT ROOTS

EPA Science Inventory

A model system was designed to measure viral uptake through the roots of plants and translocation to distal plant parts. For this study, uptake of bacteriophage f2 was measured in corn and bean plants growing in hydroponic solutions. Few phage were detected in plants with uncut r...

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

PubMed

Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

2016-11-15

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Is the detection of aquatic environmental DNA influenced by substrate type?

PubMed

Buxton, Andrew S; Groombridge, Jim J; Griffiths, Richard A

2017-01-01

The use of environmental DNA (eDNA) to assess the presence-absence of rare, cryptic or invasive species is hindered by a poor understanding of the factors that can remove DNA from the system. In aquatic systems, eDNA can be transported out either horizontally in water flows or vertically by incorporation into the sediment. Equally, eDNA may be broken down by various biotic and abiotic processes if the target organism leaves the system. We use occupancy modelling and a replicated mesocosm experiment to examine how detection probability of eDNA changes once the target species is no longer present. We hypothesise that detection probability falls faster with a sediment which has a large number of DNA binding sites such as topsoil or clay, over lower DNA binding capacity substrates such as sand. Water removed from ponds containing the target species (the great crested newt) initially showed high detection probabilities, but these fell to between 40% and 60% over the first 10 days and to between 10% and 22% by day 15: eDNA remained detectable at very low levels until day 22. Very little difference in detection was observed between the control group (no substrate) and the sand substrate. A small reduction in detection probability was observed between the control and clay substrates, but this was not significant. However, a highly significant reduction in detection probability was observed with a topsoil substrate. This result is likely to have stemmed from increased levels of PCR inhibition, suggesting that incorporation of DNA into the sentiment is of only limited importance. Surveys of aquatic species using eDNA clearly need to take account of substrate type as well as other environmental factors when collecting samples, analysing data and interpreting the results.

Multitarget stool DNA tests increases colorectal cancer screening among previously noncompliant Medicare patients

PubMed Central

Prince, Mark; Lester, Lynn; Chiniwala, Rupal; Berger, Barry

2017-01-01

AIM To determine the uptake of noninvasive multitarget stool DNA (mt-sDNA) in a cohort of colorectal cancer (CRC) screening non-compliant average-risk Medicare patients. METHODS This cross sectional primary care office-based study examined mt-sDNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-sDNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-sDNA compliance and diagnostic colonoscopy compliance on positive cases. RESULTS Over 12 mo, 77 providers ordered 393 mt-sDNA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sDNA was negative in 85.3% (296/347) and positive in 14.7% (51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer (4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas (15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage I (2) and Stage II (2), three were located in the proximal colon and one was located in the distal colon. CONCLUSION Mt-sDNA provided medical benefit to screening noncompliant Medicare population. High compliance with mt-sDNA and subsequent follow-up diagnostic colonoscopy identified patients with clinically critical advanced colorectal neoplasia. PMID:28210082

Multitarget stool DNA tests increases colorectal cancer screening among previously noncompliant Medicare patients.

PubMed

Prince, Mark; Lester, Lynn; Chiniwala, Rupal; Berger, Barry

2017-01-21

To determine the uptake of noninvasive multitarget stool DNA (mt-sDNA) in a cohort of colorectal cancer (CRC) screening non-compliant average-risk Medicare patients. This cross sectional primary care office-based study examined mt-sDNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-sDNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-sDNA compliance and diagnostic colonoscopy compliance on positive cases. Over 12 mo, 77 providers ordered 393 mt-sDNA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sDNA was negative in 85.3% (296/347) and positive in 14.7% (51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer (4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas (15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage I (2) and Stage II (2), three were located in the proximal colon and one was located in the distal colon. Mt-sDNA provided medical benefit to screening noncompliant Medicare population. High compliance with mt-sDNA and subsequent follow-up diagnostic colonoscopy identified patients with clinically critical advanced colorectal neoplasia.

Expression of insulin-like growth factor-2 receptors on EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2007-01-01

In the present study, we report the upregulation of functional IGF-2Rs in cells overexpressing growth hormone (GH). EL4 lymphoma cells stably transfected with an rGH cDNA overexpression vector (GHo) exhibited an increase in the binding of (125)I-IGF-2 with no change in the binding affinity compared to vector alone controls. An increase in the expression of the insulin-like growth factor-2 receptor (IGF-2R) in cells overexpressing GH was confirmed by Western blot analysis and IGF-2R promoter luciferase assays. EL4 cells produce insulin-like growth factor-2 (IGF-2) as detected by the reverse transcription-polymerase chain reaction (RT-PCR); however, no IGF-2 protein was detected by Western analysis. The increase in the expression of the IGF-2R resulted in greater levels of IGF-2 uptake in GHo cells compared to vector alone controls. The data suggest that one of the consequences of the overexpression of GH is an increase in the expression of the IGF-2R.

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

NASA Astrophysics Data System (ADS)

Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

2012-08-01

Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.

Structural and Mechanistic Basis of Zinc Regulation Across the E. coli Zur Regulon

PubMed Central

Gilston, Benjamin A.; Wang, Suning; Marcus, Mason D.; Canalizo-Hernández, Mónica A.; Swindell, Elden P.; Xue, Yi; Mondragón, Alfonso; O'Halloran, Thomas V.

2014-01-01

Commensal microbes, whether they are beneficial or pathogenic, are sensitive to host processes that starve or swamp the prokaryote with large fluctuations in local zinc concentration. To understand how microorganisms coordinate a dynamic response to changes in zinc availability at the molecular level, we evaluated the molecular mechanism of the zinc-sensing zinc uptake regulator (Zur) protein at each of the known Zur-regulated genes in Escherichia coli. We solved the structure of zinc-loaded Zur bound to the PznuABC promoter and show that this metalloregulatory protein represses gene expression by a highly cooperative binding of two adjacent dimers to essentially encircle the core element of each of the Zur-regulated promoters. Cooperativity in these protein-DNA interactions requires a pair of asymmetric salt bridges between Arg52 and Asp49′ that connect otherwise independent dimers. Analysis of the protein-DNA interface led to the discovery of a new member of the Zur-regulon: pliG. We demonstrate this gene is directly regulated by Zur in a zinc responsive manner. The pliG promoter forms stable complexes with either one or two Zur dimers with significantly less protein-DNA cooperativity than observed at other Zur regulon promoters. Comparison of the in vitro Zur-DNA binding affinity at each of four Zur-regulon promoters reveals ca. 10,000-fold variation Zur-DNA binding constants. The degree of Zur repression observed in vivo by comparison of transcript copy number in wild-type and Δzur strains parallels this trend spanning a 100-fold difference. We conclude that the number of ferric uptake regulator (Fur)-family dimers that bind within any given promoter varies significantly and that the thermodynamic profile of the Zur-DNA interactions directly correlates with the physiological response at different promoters. PMID:25369000

Simultaneous analysis of six aldehyde-DNA adducts in salivary DNA of nonsmokers and smokers using stable isotope dilution liquid chromatography electrospray ionization-tandem mass spectrometry.

PubMed

Li, Xiangyu; Liu, Lujuan; Wang, Hongjuan; Chen, Jian; Zhu, Beibei; Chen, Huan; Hou, Hongwei; Hu, Qingyuan

2017-08-15

A stable method, using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), to simultaneously determine six aldehyde-DNA adducts was developed and applied to the analysis of human salivary DNA samples. The detection limit of these six DNA adducts was in the range of 0.006-0.014ng/mL and that of the quantification limit was 0.017-0.026ng/mL. The intra-day and inter-day precision of all aldehyde-DNA adducts was <10%. The analysis was completed within 25min. Additionally, a noninvasive technique was used to collect the DNA samples from human saliva. The new method was successfully applied for the analysis of salivary DNA of nonsmokers and smokers. Five aldehyde-DNA adducts were detected in both smoker and nonsmoker salivary DNA, while α-Acr-dG was not detected in all the samples. Among these detected DNA adducts, no significant differences were found between smoker and nonsmoker (p>0.05). This may due to the individual detoxifying differences or environmental and endogenous exposure. Our study provides a rapid and selective method to simultaneously detect six aldehyde-DNA adducts and to assess potential DNA damage induced by aldehydes. Copyright © 2017 Elsevier B.V. All rights reserved.

Clearing muddied waters: Capture of environmental DNA from turbid waters.

PubMed

Williams, Kelly E; Huyvaert, Kathryn P; Piaggio, Antoinette J

2017-01-01

Understanding the differences in efficiencies of various methods to concentrate, extract, and amplify environmental DNA (eDNA) is vital for best performance of eDNA detection. Aquatic systems vary in characteristics such as turbidity, eDNA concentration, and inhibitor load, thus affecting eDNA capture efficiency. Application of eDNA techniques to the detection of terrestrial invasive or endangered species may require sampling at intermittent water sources that are used for drinking and cooling; these water bodies may often be stagnant and turbid. We present our best practices technique for the detection of wild pig eDNA in water samples, a protocol that will have wide applicability to the detection of elusive vertebrate species. We determined the best practice for eDNA capture in a turbid water system was to concentrate DNA from a 15 mL water sample via centrifugation, purify DNA with the DNeasy mericon Food kit, and remove inhibitors with Zymo Inhibitor Removal Technology columns. Further, we compared the sensitivity of conventional PCR to quantitative PCR and found that quantitative PCR was more sensitive in detecting lower concentrations of eDNA. We show significant differences in efficiencies among methods in each step of eDNA capture, emphasizing the importance of optimizing best practices for the system of interest.

Clearing muddied waters: Capture of environmental DNA from turbid waters

PubMed Central

Huyvaert, Kathryn P.; Piaggio, Antoinette J.

2017-01-01

Understanding the differences in efficiencies of various methods to concentrate, extract, and amplify environmental DNA (eDNA) is vital for best performance of eDNA detection. Aquatic systems vary in characteristics such as turbidity, eDNA concentration, and inhibitor load, thus affecting eDNA capture efficiency. Application of eDNA techniques to the detection of terrestrial invasive or endangered species may require sampling at intermittent water sources that are used for drinking and cooling; these water bodies may often be stagnant and turbid. We present our best practices technique for the detection of wild pig eDNA in water samples, a protocol that will have wide applicability to the detection of elusive vertebrate species. We determined the best practice for eDNA capture in a turbid water system was to concentrate DNA from a 15 mL water sample via centrifugation, purify DNA with the DNeasy mericon Food kit, and remove inhibitors with Zymo Inhibitor Removal Technology columns. Further, we compared the sensitivity of conventional PCR to quantitative PCR and found that quantitative PCR was more sensitive in detecting lower concentrations of eDNA. We show significant differences in efficiencies among methods in each step of eDNA capture, emphasizing the importance of optimizing best practices for the system of interest. PMID:28686659

Radiolabeled Rhein as Small-Molecule Necrosis Avid Agents for Imaging of Necrotic Myocardium.

PubMed

Luo, Qi; Jin, Qiaomei; Su, Chang; Zhang, Dongjian; Jiang, Cuihua; Fish, Anne Folta; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Yin, Zhiqi

2017-01-17

A rapid and accurate identification of necrotic myocardium is of great importance for diagnosis, risk stratification, clinical decision-making, and prognosis evaluation of myocardial infarction. Here, we explored technetium-99m labeled rhein derivatives for rapid imaging of the necrotic myocardium. Three hydrazinonicotinic acid-linker-rhein (HYNIC-linker-rhein) derivatives were synthesized, and then, these synthetic compounds were labeled with technetium-99m using ethylenediaminediacetic acid (EDDA) and tricine as coligands [ 99m Tc(EDDA)-HYNIC-linker-rhein]. The necrosis avidity of the three 99m Tc-labeled rhein derivatives was tested in a mouse model of ethanol-induced muscular necrosis by gamma counting, histochemical staining, and autoradiography. A lead tracer for visualization of necrotic myocardium was assessed by single photon emission computed tomography/computed tomography (SPECT/CT) imaging in a rat model with reperfused myocardial infarction. The necrosis avidity mechanism of the tracer was explored by DNA binding studies in vitro and blocking experiments in vivo. Results showed that the uptake in necrotic muscles of the three 99m Tc-compounds was higher than that in viable muscles (P < 0.001). Autoradiography and histochemical staining results were consistent with selective uptake of the radiotracer in the necrotic regions. Among the these tracers, 99m Tc(EDDA)-HYNIC-ethylenediamine-rhein [ 99m Tc(EDDA)-HYNIC-2C-rhein] displayed the best distribution profiles for imaging. The necrotic myocardium lesions were clearly visualized by SPECT/CT using 99m Tc(EDDA)-HYNIC-2C-rhein at 1 h after injection. The necrotic-to-viable myocardium and necrotic myocardium-to-blood uptake ratios of 99m Tc(EDDA)-HYNIC-2C-rhein were 4.79 and 3.02 at 1 h after injection. DNA binding studies suggested HYNIC-linker-rhein bound to DNA through intercalation. The uptake of 99m Tc(EDDA)-HYNIC-2C-rhein in necrotic muscle was significantly blocked by excessive unlabeled rhein, with 77.61% decline at 1 h after coinjection. These findings suggested 99m Tc(EDDA)-HYNIC-2C-rhein emerged as a "hot spot" imaging probe that has a potential for rapid imaging of necrotic myocardium. The necrosis avidity mechanism of 99m Tc(EDDA)-HYNIC-linker-rhein may be due to its interaction with exposed DNA in necrotic tissues.

Efficient DNA binding and nuclear uptake by distamycin derivatives conjugated to octa-arginine sequences.

PubMed

Vázquez, Olalla; Blanco-Canosa, Juan B; Vázquez, M Eugenio; Martínez-Costas, Jose; Castedo, Luis; Mascareñas, José L

2008-11-24

Efficient targeting of DNA by designed molecules requires not only careful fine-tuning of their DNA-recognition properties, but also appropriate cell internalization of the compounds so that they can reach the cell nucleus in a short period of time. Previous observations in our group on the relatively high affinity displayed by conjugates between distamycin derivatives and bZIP basic regions for A-rich DNA sites, led us to investigate whether the covalent attachment of a positively charged cell-penetrating peptide to a distamycin-like tripyrrole might yield high affinity DNA binders with improved cell internalization properties. Our work has led to the discovery of synthetic tripyrrole-octa-arginine conjugates that are capable of targeting specific DNA sites that contain A-rich tracts with low nanomolar affinity; they simultaneously exhibit excellent membrane and nuclear translocation properties in living HeLa cells.

Visual Servoing for Optimization of Anticancer Drug Uptake in Human Breast Cancer Cells

DTIC Science & Technology

2000-09-01

successfully obtained new DOE Medical Applications Program funding for this research (included in Appendix G: Automated Imaging System for Guiding Antisense ...Guiding Antisense Compounds to Specific mRNVA targets in Living Cells ) that will support this integration and development work with Dr. Parvin and Deep...a DNA and RNA binding fluorescence probe with a very different emission wavelengths, depending on whether it is bound to DNA or RNA ). Cells were then

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Jianmin; Weaver, L.M.; Herrmann, K.M.

A cDNA for potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, encodes a 56 KD polypeptide whose amino terminus resembles a chloroplast transit sequence. The cDNA was placed downstream of the phage T7 polymerase recognition sequence in plasmid pGEM-3Z. DNA of the resulting plasmid pGEM-DWZ directed T7 polymerase to synthesize potato DAHP synthase mRNA in vitro. The mRNA was used in wheat germ and rabbit reticulocyte lysates for the synthesis of {sup 35}S-labeled pro-DAHP synthase. The predominant translation product is a 59 KD polypeptide that can be immunoprecipitated by rabbit polyclonal antibodies raised againstmore » the 53 KD DAHP synthase purified from potato tubers. Isolated spinach chloroplasts process the 59 KD pro-DAHP synthase to a 50 KD polypeptide. The processed polypeptide is protected from protease degradation, suggesting uptake of the enzyme into the cell organelle. Fractionation of reisolated chloroplasts after import of pro-DAHP synthase showed mature enzyme in the stroma. The uptake and processing of DAHP synthase is inhibited by antibodies raised against the mature enzyme. Our results are consistent with the assumption that potato contains a nuclear DNA encoded DAHP synthase that is synthesized as a proenzyme and whose mature form resides in the chloroplasts. Our data provide further evidence that green plants synthesize aromatic amino acids in plastids.« less

Well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length.

PubMed

Tang, Rupei; Palumbo, R Noelle; Nagarajan, Lakshmi; Krogstad, Emily; Wang, Chun

2010-03-03

The development of safe and efficient polymer carriers for DNA vaccine delivery requires mechanistic understanding of structure-function relationship of the polymer carriers and their interaction with antigen-presenting cells. Here we have synthesized a series of diblock copolymers with well-defined chain-length using atom transfer radical polymerization and characterized the influence of polycation chain-length on the physico-chemical properties of the polymer/DNA complexes as well as the interaction with dendritic cells. The copolymers consist of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (PAEM) block. The average degree of polymerization (DP) of the PAEM block was varied among 19, 39, and 75, with nearly uniform distribution. With increasing PAEM chain-length, polyplexes formed by the diblock copolymers and plasmid DNA had smaller average particle size and showed higher stability against electrostatic destabilization by salt and heparin. The polymers were not toxic to mouse dendritic cells (DCs) and only displayed chain-length-dependent toxicity at a high concentration (1mg/mL). In vitro gene transfection efficiency and polyplex uptake in DCs were also found to correlate with chain-length of the PAEM block with the longer polymer chain favoring transfection and cellular uptake. The polyplexes induced a modest up-regulation of surface markers for DC maturation that was not significantly dependent on PAEM chain-length. Finally, the polyplex prepared from the longest PAEM block (DP of 75) achieved an average of 20% enhancement over non-condensed anionic dextran in terms of uptake by DCs in the draining lymph nodes 24h after subcutaneous injection into mice. Insights gained from studying such structurally well-defined polymer carriers and their interaction with dendritic cells may contribute to improved design of practically useful DNA vaccine delivery systems. Copyright 2009 Elsevier B.V. All rights reserved.

Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors.

PubMed

Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

2010-07-02

Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

PubMed

Johnston, Christopher D; Skeete, Chelsey A; Fomenkov, Alexey; Roberts, Richard J; Rittling, Susan R

2017-01-01

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia

PubMed Central

Skeete, Chelsey A.; Fomenkov, Alexey; Roberts, Richard J.; Rittling, Susan R.

2017-01-01

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease. PMID:28934361

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium.

PubMed

Pan, Xiao-Ben; Wei, Lai; Han, Jin-Chao; Gao, Yan

2008-01-01

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection. (c) 2007 Wiley-Liss, Inc.

DNA-based species detection capabilities using laser transmission spectroscopy

PubMed Central

Mahon, A. R.; Barnes, M. A.; Li, F.; Egan, S. P.; Tanner, C. E.; Ruggiero, S. T.; Feder, J. L.; Lodge, D. M.

2013-01-01

Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel (Dreissena bugensis) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications. PMID:23015524

"Signal-on" photoelectrochemical biosensor for sensitive detection of human T-Cell lymphotropic virus type II DNA: dual signal amplification strategy integrating enzymatic amplification with terminal deoxynucleotidyl transferase-mediated extension.

PubMed

Shen, Qingming; Han, Li; Fan, Gaochao; Zhang, Jian-Rong; Jiang, Liping; Zhu, Jun-Jie

2015-01-01

A novel "signal-on" photoelectrochemical (PEC) biosensor for sensitive detection of human T-cell lymphotropic virus type II (HTLV-II) DNA was developed on the basis of enzymatic amplification coupled with terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. The intensity of the photocurrent signal was proportional to the concentration of the HTLV-II DNA-target DNA (tDNA) by dual signal amplification. In this protocol, GR-CdS:Mn/ZnS nanocomposites were used as photoelectric conversion material, while pDNA was used as the tDNA recognizing unit. Moreover, the TdT-mediated extension and the enzymatic signal amplification technique were used to enhance the sensitivity of detection. Using this novel dual signal amplification strategy, the prototype of PEC DNA sensor can detect as low as ∼0.033 fM of HTLV-II DNA with a linear range of 0.1-5000 fM, with excellent differentiation ability even for single-base mismatches. This PEC DNA assay opens a promising platform to detect various DNA targets at ultralow levels for early diagnoses of different diseases.

Detection of an endangered aquatic heteropteran using environmental DNA in a wetland ecosystem

PubMed Central

Katano, Izumi; Sakata, Yusuke; Souma, Rio; Kosuge, Toshihiro; Nagano, Mariko; Ikeda, Kousuke; Yano, Koki; Tojo, Koji

2017-01-01

The use of environmental DNA (eDNA) has recently been employed to evaluate the distribution of various aquatic macroorganisms. Although this technique has been applied to a broad range of taxa, from vertebrates to invertebrates, its application is limited for aquatic insects such as aquatic heteropterans. Nepa hoffmanni (Heteroptera: Nepidae) is a small (approx. 23 mm) aquatic heteropteran that inhabits wetlands, can be difficult to capture and is endangered in Japan. The molecular tool eDNA was used to evaluate the species distribution of N. hoffmanni in comparison to that determined using hand-capturing methods in two regions of Japan. The eDNA of N. hoffmanni was detected at nearly all sites (10 eDNA-detected sites out of 14 sites), including sites where N. hoffmanni was not captured by hand (five eDNA-detected sites out of six captured sites). Thus, this species-specific eDNA technique can be applied to detect small, sparsely distributed heteropterans in wetland ecosystems. In conclusion, eDNA could be a valuable technique for the detection of aquatic insects inhabiting wetland habitats, and could make a significant contribution to providing distribution data necessary to species conservation. PMID:28791177

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

PubMed

Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

2018-01-01

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

A universal DNA-based protein detection system.

PubMed

Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

2013-09-25

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

A Universal DNA-Based Protein Detection System

PubMed Central

Tran, Thua N. N.; Cui, Jinhui; Hartman, Mark R.; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C.; Lis, John T.; Cui, Haixin; Luo, Dan

2014-01-01

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability. PMID:23978265

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Making sense of the noise: The effect of hydrology on silver carp eDNA detection in the Chicago area waterway system.

PubMed

Song, Jeffery W; Small, Mitchell J; Casman, Elizabeth A

2017-12-15

Environmental DNA (eDNA) sampling is an emerging tool for monitoring the spread of aquatic invasive species. One confounding factor when interpreting eDNA sampling evidence is that eDNA can be present in the water in the absence of living target organisms, originating from excreta, dead tissue, boats, or sewage effluent, etc. In the Chicago Area Waterway System (CAWS), electric fish dispersal barriers were built to prevent non-native Asian carp species from invading Lake Michigan, and yet Asian carp eDNA has been detected above the barriers sporadically since 2009. In this paper the influence of stream flow characteristics in the CAWS on the probability of invasive Asian carp eDNA detection in the CAWS from 2009 to 2012 was examined. In the CAWS, the direction of stream flow is mostly away from Lake Michigan, though there are infrequent reversals in flow direction towards Lake Michigan during dry spells. We find that the flow reversal volume into the Lake has a statistically significant positive relationship with eDNA detection probability, while other covariates, like gage height, precipitation, season, water temperature, dissolved oxygen concentration, pH and chlorophyll concentration do not. This suggests that stream flow direction is highly influential on eDNA detection in the CAWS and should be considered when interpreting eDNA evidence. We also find that the beta-binomial regression model provides a stronger fit for eDNA detection probability compared to a binomial regression model. This paper provides a statistical modeling framework for interpreting eDNA sampling evidence and for evaluating covariates influencing eDNA detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Photoactive roadways: Determination of CO, NO and VOC uptake coefficients and photolabile side product yields on TiO2 treated asphalt and concrete

NASA Astrophysics Data System (ADS)

Toro, C.; Jobson, B. T.; Haselbach, L.; Shen, S.; Chung, S. H.

2016-08-01

This work reports uptake coefficients and by-product yields of ozone precursors onto two photocatalytic paving materials (asphalt and concrete) treated with a commercial TiO2 surface application product. The experimental approach used a continuously stirred tank reactor (CSTR) and allowed for testing large samples with the same surface morphology encountered with real urban surfaces. The measured uptake coefficient (γgeo) and surface resistances are useful for parametrizing dry deposition velocities in air quality model evaluation of the impact of photoactive surfaces on urban air chemistry. At 46% relative humidity, the surface resistance to NO uptake was ∼1 s cm-1 for concrete and ∼2 s cm-1 for a freshly coated older roadway asphalt sample. HONO and NO2 were detected as side products from NO uptake to asphalt, with NO2 molar yields on the order of 20% and HONO molar yields ranging between 14 and 33%. For concrete samples, the NO2 molar yields increased with the increase of water vapor, ranging from 1% to 35% and HONO was not detected as a by-product. Uptake of monoaromatic VOCs to the asphalt sample set displayed a dependence on the compound vapor pressure, and was influenced by competitive adsorption from less volatile VOCs. Formaldehyde and acetaldehyde were detected as byproducts, with molar yields ranging from 5 to 32%.

Using occupancy modelling to compare environmental DNA to traditional field methods for regional-scale monitoring of an endangered aquatic species.

PubMed

Schmelzle, Molly C; Kinziger, Andrew P

2016-07-01

Environmental DNA (eDNA) monitoring approaches promise to greatly improve detection of rare, endangered and invasive species in comparison with traditional field approaches. Herein, eDNA approaches and traditional seining methods were applied at 29 research locations to compare method-specific estimates of detection and occupancy probabilities for endangered tidewater goby (Eucyclogobius newberryi). At each location, multiple paired seine hauls and water samples for eDNA analysis were taken, ranging from two to 23 samples per site, depending upon habitat size. Analysis using a multimethod occupancy modelling framework indicated that the probability of detection using eDNA was nearly double (0.74) the rate of detection for seining (0.39). The higher detection rates afforded by eDNA allowed determination of tidewater goby occupancy at two locations where they have not been previously detected and at one location considered to be locally extirpated. Additionally, eDNA concentration was positively related to tidewater goby catch per unit effort, suggesting eDNA could potentially be used as a proxy for local tidewater goby abundance. Compared to traditional field sampling, eDNA provided improved occupancy parameter estimates and can be applied to increase management efficiency across a broad spatial range and within a diversity of habitats. © 2015 John Wiley & Sons Ltd.

Quartz crystal microbalance detection of DNA single-base mutation based on monobase-coded cadmium tellurium nanoprobe.

PubMed

Zhang, Yuqin; Lin, Fanbo; Zhang, Youyu; Li, Haitao; Zeng, Yue; Tang, Hao; Yao, Shouzhuo

2011-01-01

A new method for the detection of point mutation in DNA based on the monobase-coded cadmium tellurium nanoprobes and the quartz crystal microbalance (QCM) technique was reported. A point mutation (single-base, adenine, thymine, cytosine, and guanine, namely, A, T, C and G, mutation in DNA strand, respectively) DNA QCM sensor was fabricated by immobilizing single-base mutation DNA modified magnetic beads onto the electrode surface with an external magnetic field near the electrode. The DNA-modified magnetic beads were obtained from the biotin-avidin affinity reaction of biotinylated DNA and streptavidin-functionalized core/shell Fe(3)O(4)/Au magnetic nanoparticles, followed by a DNA hybridization reaction. Single-base coded CdTe nanoprobes (A-CdTe, T-CdTe, C-CdTe and G-CdTe, respectively) were used as the detection probes. The mutation site in DNA was distinguished by detecting the decreases of the resonance frequency of the piezoelectric quartz crystal when the coded nanoprobe was added to the test system. This proposed detection strategy for point mutation in DNA is proved to be sensitive, simple, repeatable and low-cost, consequently, it has a great potential for single nucleotide polymorphism (SNP) detection. 2011 © The Japan Society for Analytical Chemistry

Factors influencing detection of eDNA from a stream-dwelling amphibian

USGS Publications Warehouse

Pilliod, David S.; Goldberg, Caren S.; Arkle, Robert S.; Waits, Lisette P.

2013-01-01

Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream-dwelling salamanders and quantitative PCR (qPCR) analysis, we conducted three experiments to assess eDNA: (i) production rate; (ii) persistence time under different temperature and light conditions; and (iii) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 77 ng eDNA/h for 2 h, after which production either slowed considerably or began to equilibrate with degradation. eDNA in both full-sun and shaded treatments degraded exponentially to 2) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 h after introduction, reaching 0.0022 ng/L. Within 1 h of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.

Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization

NASA Astrophysics Data System (ADS)

Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E.; Ding, Zhong-Tao

2015-02-01

In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs.

Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals.

PubMed

Foote, Andrew D; Thomsen, Philip Francis; Sveegaard, Signe; Wahlberg, Magnus; Kielgast, Jos; Kyhn, Line A; Salling, Andreas B; Galatius, Anders; Orlando, Ludovic; Gilbert, M Thomas P

2012-01-01

The exploitation of non-invasive samples has been widely used in genetic monitoring of terrestrial species. In aquatic ecosystems, non-invasive samples such as feces, shed hair or skin, are less accessible. However, the use of environmental DNA (eDNA) has recently been shown to be an effective tool for genetic monitoring of species presence in freshwater ecosystems. Detecting species in the marine environment using eDNA potentially offers a greater challenge due to the greater dilution, amount of mixing and salinity compared with most freshwater ecosystems. To determine the potential use of eDNA for genetic monitoring we used specific primers that amplify short mitochondrial DNA sequences to detect the presence of a marine mammal, the harbor porpoise, Phocoena phocoena, in a controlled environment and in natural marine locations. The reliability of the genetic detections was investigated by comparing with detections of harbor porpoise echolocation clicks by static acoustic monitoring devices. While we were able to consistently genetically detect the target species under controlled conditions, the results from natural locations were less consistent and detection by eDNA was less successful than acoustic detections. However, at one site we detected long-finned pilot whale, Globicephala melas, a species rarely sighted in the Baltic. Therefore, with optimization aimed towards processing larger volumes of seawater this method has the potential to compliment current visual and acoustic methods of species detection of marine mammals.

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

PubMed

Vorsters, A; Van den Bergh, J; Micalessi, I; Biesmans, S; Bogers, J; Hens, A; De Coster, I; Ieven, M; Van Damme, P

2014-11-01

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

PubMed

Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Detection of influenza A virus using carbon nanotubes field effect transistor based DNA sensor

NASA Astrophysics Data System (ADS)

Tran, Thi Luyen; Nguyen, Thi Thuy; Huyen Tran, Thi Thu; Chu, Van Tuan; Thinh Tran, Quang; Tuan Mai, Anh

2017-09-01

The carbon nanotubes field effect transistor (CNTFET) based DNA sensor was developed, in this paper, for detection of influenza A virus DNA. Number of factors that influence the output signal and analytical results were investigated. The initial probe DNA, decides the available DNA strands on CNTs, was 10 μM. The hybridization time for defined single helix was 120 min. The hybridization temperature was set at 30 °C to get a net change in drain current of the DNA sensor without altering properties of any biological compounds. The response time of the DNA sensor was less than one minute with a high reproducibility. In addition, the DNA sensor has a wide linear detection range from 1 pM to 10 nM, and a very low detection limit of 1 pM. Finally, after 7-month storage in 7.4 pH buffer, the output signal of DNA sensor recovered 97%.

Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Y.; Dunn, J.; Gao, S.

2008-10-31

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing asmore » little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.« less

Cutaneous gallium uptake in patients with AIDS with mycobacterium avium-intracellulare septicemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allwright, S.J.; Chapman, P.R.; Antico, V.F.

1988-07-01

Gallium imaging is increasingly being used for the early detection of complications in patients with AIDS. A 26-year-old homosexual man who was HIV antibody positive underwent gallium imaging for investigation of possible Pneumocystis carinii pneumonia. Widespread cutaneous focal uptake was seen, which was subsequently shown to be due to mycobacterium avium-intracellulare (MAI) septicemia. This case demonstrates the importance of whole body imaging rather than imaging target areas only, the utility of gallium imaging in aiding the early detection of clinically unsuspected disease, and shows a new pattern of gallium uptake in disseminated MAI infection.

Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevices.

PubMed

Diao, Wei; Tang, Min; Ding, Shijia; Li, Xinmin; Cheng, Wenbin; Mo, Fei; Yan, Xiaoyu; Ma, Hongmin; Yan, Yurong

2018-02-15

Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Studies on the mechanism of benzene toxicity.

PubMed Central

Snyder, R; Dimitriadis, E; Guy, R; Hu, P; Cooper, K; Bauer, H; Witz, G; Goldstein, B D

1989-01-01

Using the 59Fe uptake method of Lee et al. it was shown that erythropoiesis in female mice was inhibited following IP administration of benzene, hydroquinone, p-benzoquinone, and muconaldehyde. Toluene protected against the effects of benzene. Coadministration of phenol plus either hydroquinone or catechol resulted in greatly increased toxicity. The combination of metabolites most effective in reducing iron uptake was hydroquinone plus muconaldehyde. We have also shown that treating animals with benzene leads to the formation of adducts of bone marrow DNA as measured by the 32P-postlabeling technique. PMID:2792049

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

PubMed Central

Barakat, Tahsin Stefan; Gribnau, Joost

2014-01-01

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

PubMed

Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

2016-07-15

A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Epstein-Barr virus DNA is detected in peripheral blood mononuclear cells of EBV-seronegative infants with infectious mononucleosis-like symptoms.

PubMed

Ikuta, Kazufumi; Saiga, Kyoko; Deguchi, Masanori; Sairenji, Takeshi

2003-01-01

We demonstrated Epstein-Barr virus (EBV) DNA in peripheral blood mononuclear cells (PBMCs) from infants with infectious mononucleosis- (IM) like symptoms. Thirteen of the 17 patients did not have EBV antibodies; however, EBV DNA was detected in 8 PBMC from the 13 seronegative patients by PCR. The 4 patients were retested in 6-12 months later. Three patients were still seronegative; however EBV DNA wasnot detected. One patient seroconverted and EBV DNA could still be detected. The transcript of EBNA1 was detected in one patient, but neither EBNA2 nor LMP2A were detected in all PBMC from the 4 tested patients. Type 1 EBV DNA was detected in 5 PBMC of 7 tested patients, and type 2 EBV DNA was detected in type 1 positive PBMC of one patient as well. The IL-1 beta polymorphism that is reported to be one of the immunological factors of EBV seronegativity revealed no difference in IM-like patients. These results indicated that EBV infection occurs in EBV-seronegative IM-like infants; however, the modes of infection are clearly different from IM.

Functional involvement of the organic cation transporter 2 (rOct2) in the renal uptake of organic cations in rats.

PubMed

Umehara, K-I; Iwatsubo, T; Noguchi, K; Kamimura, H

2008-01-01

This study examined the contribution made by organic cation transporters (hOCT/rOct) to the saturable component of the renal uptake of 1-methyl-4-phenylpyridinium, tetraethylammonium (TEA), cimetidine and metformin into rOct2-expressing HEK293 cells and rat kidney slices. All the test compounds accumulated in the rat kidney slices in a carrier-mediated manner. The Michaelis- Menten constant (K(m)) values for saturable uptake of TEA, cimetidine and metformin into rat kidney slices were relatively comparable with those for the rOct2-expressing HEK293 cells. In addition, the relative uptake activity values of TEA, cimetidine and metformin in rat kidney slices were similar to those in rOct2-expressing HEK293 cells. This suggests that the saturable components involved in the renal uptake of TEA, cimetidine and metformin are mediated mainly by rOct2. The saturable uptake profile of cationic compounds into rat kidney can be evaluated in both cDNA-expressing cells and rat kidney slices, as well as the transporter expression pattern. This approach can also be used to estimate the saturable uptake mechanism of cationic compounds into the human kidney when human kidney slices and hOCT2-expressing cells are used.

Ultrasensitive photoelectrochemical biosensor for the detection of HTLV-I DNA: A cascade signal amplification strategy integrating λ-exonuclease aided target recycling with hybridization chain reaction and enzyme catalysis.

PubMed

Shi, Xiao-Mei; Fan, Gao-Chao; Tang, Xueying; Shen, Qingming; Zhu, Jun-Jie

2018-06-30

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h 1 DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h 1 DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h 2 DNA and h 3 DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.

PubMed

Eichmiller, Jessica J; Miller, Loren M; Sorensen, Peter W

2016-01-01

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2-0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies. © 2015 John Wiley & Sons Ltd.

Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

NASA Astrophysics Data System (ADS)

Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

2013-11-01

A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells. Electronic supplementary information (ESI) available: http://janus.northwestern.edu/wololab/auxiliary/supplementary_data_2013.docx. See DOI: 10.1039/c3nr02203j

Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules.

PubMed

Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; Khairil Mokhtar, Nur Fadhilah; El Sheikha, Aly Farag

2018-03-05

The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared. A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA. The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Toward radioguided surgery with β- decays: uptake of a somatostatin analogue, DOTATOC, in meningioma and high-grade glioma.

PubMed

Collamati, Francesco; Pepe, Alessandra; Bellini, Fabio; Bocci, Valerio; Chiodi, Giacomo; Cremonesi, Marta; De Lucia, Erika; Ferrari, Mahila E; Frallicciardi, Paola M; Grana, Chiara M; Marafini, Michela; Mattei, Ilaria; Morganti, Silvio; Patera, Vincenzo; Piersanti, Luca; Recchia, Luigi; Russomando, Andrea; Sarti, Alessio; Sciubba, Adalberto; Senzacqua, Martina; Solfaroli Camillocci, Elena; Voena, Cecilia; Pinci, Davide; Faccini, Riccardo

2015-01-01

A novel radioguided surgery (RGS) technique for cerebral tumors using β(-) radiation is being developed. Checking for a radiotracer that can deliver a β(-) emitter to the tumor is a fundamental step in the deployment of such a technique. This paper reports a study of the uptake of (90)Y-DOTATOC in meningiomas and high-grade gliomas (HGGs) and a feasibility study of the RGS technique in these types of tumor. Estimates were performed assuming the use of a β(-) probe under development with a sensitive area 2.55 mm in radius to detect 0.1-mL residuals. Uptake and background from healthy tissues were estimated on (68)Ga-DOTATOC PET scans of 11 meningioma patients and 12 HGG patients. A dedicated statistical analysis of the DICOM images was developed and validated. The feasibility study was performed using full simulation of emission and detection of the radiation, accounting for the measured uptake and background rate. All meningioma patients but one with an atypical extracranial tumor showed high uptake of DOTATOC. In terms of feasibility of the RGS technique, we estimated that by administering a 3 MBq/kg activity of radiotracer, the time needed to detect a 0.1-mL remnant with 5% false-negative and 1% false-positive rates is less than 1 s. Actually, to achieve a detection time of 1 s the required activities to administer were as low as 0.2-0.5 MBq/kg in many patients. In HGGs, the uptake was lower than in meningiomas, but the tumor-to-nontumor ratio was higher than 4, which implies that the tracer can still be effective for RGS. It was estimated that by administering 3 mBq/kg of radiotracer, the time needed to detect a 0.1-mL remnant is less than 6 s, with the exception of the only oligodendroma in the sample. Uptake of (90)Y-DOTATOC in meningiomas was high in all studied patients. Uptake in HGGs was significantly worse than in meningiomas but was still acceptable for RGS, particularly if further research and development are done to improve the performance of the β(-) probe. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma.

PubMed

Tang, Dong-ling; Li, Yan; Zhou, Xin; Li, Xia; Zheng, Fang

2009-05-01

To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment. Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Laser desorption mass spectrometry for biomolecule detection and its applications

NASA Astrophysics Data System (ADS)

Winston Chen, C. H.; Sammartano, L. J.; Isola, N. R.; Allman, S. L.

2001-08-01

During the past few years, we developed and used laser desorption mass spectrometry for biomolecule detections. Matrix-assisted laser desorption/ionization (MALDI) was successfully used to detect DNA fragments with the size larger than 3000 base pairs. It was also successfully used to sequence DNA with both enzymatic and chemical degradation methods to produce DNA ladders. We also developed MALDI with fragmentation for direct DNA sequencing for short DNA probes. Since laser desorption mass spectrometry for DNA detection has the advantages of fast speed and no need of labeling, it has a great potential for molecular diagnosis for disease and person identification by DNA fingerprinting. We applied laser desorption mass spectrometry to succeed in the diagnosis of cystic fibrosis and several other nerve degenerative diseases such as Huntington's disease. We also succeeded in demonstrating DNA typing for forensic applications.

Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis.

PubMed Central

Chamberland, S; Bayer, A S; Schollaardt, T; Wong, S A; Bryan, L E

1989-01-01

Mechanisms of resistance to quinolones were characterized in Pseudomonas aeruginosa strains isolated after Tn5 insertional mutagenesis and in resistant strains that emerged during pefloxacin therapy of experimental aortic endocarditis. Quinolone resistance achieved in in vitro-selected mutants Qr-1 and Qr-2 was associated with cross-resistance to several groups of antimicrobial agents, including beta-lactams, tetracycline, and chloramphenicol. A significant reduction of norfloxacin uptake was also observed. After ether permeabilization of the cells, DNA synthesis of these two isolates was as susceptible to norfloxacin as DNA synthesis of the parent strain (PAO1). These results indicate that alteration of outer membrane permeability is the primary determinant of resistance in these isolates. This altered cell permeability was correlated with reduction of outer membrane protein G (25.5 kilodaltons) and loss of a 40-kilodalton outer membrane protein in strain Qr-1. Resistance to quinolones that emerged during experimental endocarditis therapy was associated with both modification of outer membrane permeability (decreased uptake of norfloxacin) and decreased susceptibility of DNA synthesis to norfloxacin. Resistance was limited to quinolones and chloramphenicol. For these strains, norfloxacin inhibitory doses (50%) for DNA synthesis were identical to the drug MICs, suggesting that despite the identification of a permeability change, perhaps due to changes of lipopolysaccharide, the alteration of the quinolone intracellular target(s) susceptibility constitutes the primary determinant of resistance. Also, two distinct levels of norfloxacin resistance of DNA synthesis were found in these isolates, indicating that at least two distinct alterations of the drug target(s) are possible in P. aeruginosa. Images PMID:2502066

Binding of the Zn2+ ion to ferric uptake regulation protein from E. coli and the competition with Fe2+ binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur

NASA Astrophysics Data System (ADS)

Jabour, Salih; Hamed, Mazen Y.

2009-04-01

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn2+ ions were added in two steps. The first step involved the binding of one Zn2+ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 Å and metal-oxygen distance of 3.1-3.2 Å. The second Zn2+ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn2+ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe2+, when added to the complex consisting of 2Zn2+/Fur dimer/DNA, replaced the Zn2+ ion in the zinc site and when a second Fe2+ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn2+ ion in the Fur dimer binding of DNA in its repressor activity.

The Relationship between the Distribution of Common Carp and Their Environmental DNA in a Small Lake

PubMed Central

Eichmiller, Jessica J.; Bajer, Przemyslaw G.; Sorensen, Peter W.

2014-01-01

Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome b gene of the common carp (Cyprinus carpio or ‘carp’), an ubiquitous invasive fish, was developed and used to measure eDNA in Lake Staring (MN, USA), in which both the density of carp and their distribution have been closely monitored for several years. Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart. PMID:25383965

The relationship between the distribution of common carp and their environmental DNA in a small lake.

PubMed

Eichmiller, Jessica J; Bajer, Przemyslaw G; Sorensen, Peter W

2014-01-01

Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome b gene of the common carp (Cyprinus carpio or 'carp'), an ubiquitous invasive fish, was developed and used to measure eDNA in Lake Staring (MN, USA), in which both the density of carp and their distribution have been closely monitored for several years. Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart.

Model-based analysis of costs and outcomes of non-invasive prenatal testing for Down's syndrome using cell free fetal DNA in the UK National Health Service.

PubMed

Morris, Stephen; Karlsen, Saffron; Chung, Nancy; Hill, Melissa; Chitty, Lyn S

2014-01-01

Non-invasive prenatal testing (NIPT) for Down's syndrome (DS) using cell free fetal DNA in maternal blood has the potential to dramatically alter the way prenatal screening and diagnosis is delivered. Before NIPT can be implemented into routine practice, information is required on its costs and benefits. We investigated the costs and outcomes of NIPT for DS as contingent testing and as first-line testing compared with the current DS screening programme in the UK National Health Service. We used a pre-existing model to evaluate the costs and outcomes associated with NIPT compared with the current DS screening programme. The analysis was based on a hypothetical screening population of 10,000 pregnant women. Model inputs were taken from published sources. The main outcome measures were number of DS cases detected, number of procedure-related miscarriages and total cost. At a screening risk cut-off of 1∶150 NIPT as contingent testing detects slightly fewer DS cases, has fewer procedure-related miscarriages, and costs the same as current DS screening (around UK£280,000) at a cost of £500 per NIPT. As first-line testing NIPT detects more DS cases, has fewer procedure-related miscarriages, and is more expensive than current screening at a cost of £50 per NIPT. When NIPT uptake increases, NIPT detects more DS cases with a small increase in procedure-related miscarriages and costs. NIPT is currently available in the private sector in the UK at a price of £400-£900. If the NHS cost was at the lower end of this range then at a screening risk cut-off of 1∶150 NIPT as contingent testing would be cost neutral or cost saving compared with current DS screening. As first-line testing NIPT is likely to produce more favourable outcomes but at greater cost. Further research is needed to evaluate NIPT under real world conditions.

Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

PubMed

Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

2013-01-01

A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA detection.

PubMed

He, Yi; Huang, Guangming; Cui, Hua

2013-11-13

It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

CAN ULTRASOUND ENABLE EFFICIENT INTRACELLULAR UPTAKE OF MOLECULES? A RETROSPECTIVE LITERATURE REVIEW AND ANALYSIS

PubMed Central

LIU, YING; YAN, JING; PRAUSNITZ, MARK R.

2012-01-01

Most applications of therapeutic ultrasound (US) for intracellular delivery of drugs, proteins, DNA/ RNA and other compounds would benefit from efficient uptake of these molecules into large numbers of cells without killing cells in the process. In this study we tested the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro. A search of the literature for studies with quantitative data on uptake and viability yielded 26 published papers containing 898 experimental data points. Analysis of these studies showed that just 7.7% of the data points corresponded to relatively efficient uptake (>50% of cells exhibiting uptake). Closer examination of the data showed that use of Definity US contrast agent (as opposed to Optison) and elevated sonication temperature at 37°C (as opposed to room temperature) were associated with high uptake, which we further validated through independent experiments carried out in this study. Although these factors contributed to high uptake, almost all data with efficient uptake were from studies that had not accounted for lysed cells when determining cell viability. Based on retrospective analysis of the data, we showed that not accounting for lysed cells can dramatically increase the calculated uptake efficiency. We further argue that if all the data considered in this study were re-analyzed to account for lysed cells, there would be essentially no data with efficient uptake. We therefore conclude that the literature does not support the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro, which poses a challenge to future applications of US that require efficient intracellular delivery. PMID:22425381

A gold@polydopamine core-shell nanoprobe for long-term intracellular detection of microRNAs in differentiating stem cells.

PubMed

Choi, Chun Kit K; Li, Jinming; Wei, Kongchang; Xu, Yang J; Ho, Lok Wai C; Zhu, Meiling; To, Kenneth K W; Choi, Chung Hang J; Bian, Liming

2015-06-17

The capability of monitoring the differentiation process in living stem cells is crucial to the understanding of stem cell biology and the practical application of stem-cell-based therapies, yet conventional methods for the analysis of biomarkers related to differentiation require a large number of cells as well as cell lysis. Such requirements lead to the unavoidable loss of cell sources and preclude real-time monitoring of cellular events. In this work, we report the detection of microRNAs (miRNAs) in living human mesenchymal stem cells (hMSCs) by using polydopamine-coated gold nanoparticles (Au@PDA NPs). The PDA shell facilitates the immobilization of fluorescently labeled hairpin DNA strands (hpDNAs) that can recognize specific miRNA targets. The gold core and PDA shell quench the fluorescence of the immobilized hpDNAs, and subsequent binding of the hpDNAs to the target miRNAs leads to their dissociation from Au@PDA NPs and the recovery of fluorescence signals. Remarkably, these Au@PDA-hpDNA nanoprobes can naturally enter stem cells, which are known for their poor transfection efficiency, without the aid of transfection agents. Upon cellular uptake of these nanoprobes, we observe intense and time-dependent fluorescence responses from two important osteogenic marker miRNAs, namely, miR-29b and miR-31, only in hMSCs undergoing osteogenic differentiation and living primary osteoblasts but not in undifferentiated hMSCs and 3T3 fibroblasts. Strikingly, our nanoprobes can afford long-term tracking of miRNAs (5 days) in the differentiating hMSCs without the need of continuously replenishing cell culture medium with fresh nanoprobes. Our results demonstrate the capability of our Au@PDA-hpDNA nanoprobes for monitoring the differentiation status of hMSCs (i.e., differentiating versus undifferentiated) via the detection of specific miRNAs in living stem cells. Our nanoprobes show great promise in the investigation of the long-term dynamics of stem cell differentiation, identification and isolation of specific cell types, and high-throughput drug screening.

An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

PubMed

Chen, Jia; Huang, Yong; Vdovenko, Marina; Sakharov, Ivan Yu; Su, Guifa; Zhao, Shulin

2015-06-01

An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

PubMed Central

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2008-01-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

Increased choline uptake in macrophages and prostate cancer cells does not allow for differentiation between benign and malignant prostate pathologies.

PubMed

Schwarz, Timo; Seidl, Christof; Schiemann, Matthias; Senekowitsch-Schmidtke, Reingard; Krause, Bernd Joachim

2016-06-01

Inflammatory cells may contribute to the choline uptake in different prostate pathologies. The aim of this study was (i) to assess if inflammatory cells incorporate choline and (ii) to potentially detect differences compared to FDG uptake. Therefore we investigated the uptake of [(3)H]choline and [(18)F]FDG in human prostate carcinoma cells and human inflammatory cells. Macrophages were cultured from isolated mononuclear cells, gained by density gradient centrifugation of human buffy coats. T-lymphocytes, B-lymphocytes and granulocytes were enriched by density gradient centrifugation before cell sorting by means of flow cytometry was performed. [(3)H]choline and [(18)F]FDG uptake of isolated inflammatory cells as well as of LNCaP and PC-3 human prostate carcinoma cells was assessed simultaneously in dual tracer uptake experiments. Macrophages showed highest [(3)H]choline and [(18)F]FDG uptake compared to the tracer uptake rates of leukocytes. [(3)H]choline uptake of macrophages was in the same range as in prostate cancer cells. Lipopolysaccharide stimulation of macrophages resulted in an increase of [(18)F]FDG uptake in macrophages, but not in an increased [(3)H]choline uptake. The high [(3)H]choline uptake in macrophages may be a source of false-positive PET results in diagnosis of prostate cancer by choline-PET/CT. As already known from FDG-PET, discrimination between tumor and inflammation in prostate cancer patients is not possible via choline-PET. The application of choline-PET for reliable primary prostate cancer detection and delineation has to be queried. Copyright © 2016 Elsevier Inc. All rights reserved.

Critical considerations for the application of environmental DNA methods to detect aquatic species

USGS Publications Warehouse

Goldberg, Caren S.; Turner, Cameron R.; Deiner, Kristy; Klymus, Katy E.; Thomsen, Philip Francis; Murphy, Melanie A.; Spear, Stephen F.; McKee, Anna; Oyler-McCance, Sara J.; Cornman, Robert S.; Laramie, Matthew B.; Mahon, Andrew R.; Lance, Richard F.; Pilliod, David S.; Strickler, Katherine M.; Waits, Lisette P.; Fremier, Alexander K.; Takahara, Teruhiko; Herder, Jelger E.; Taberlet, Pierre

2016-01-01

Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed.Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms.Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA.We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

PubMed

Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

2016-12-15

In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of highly sensitive electrochemical genosensor based on multiwalled carbon nanotubes-chitosan-bismuth and lead sulfide nanoparticles for the detection of pathogenic Aeromonas.

PubMed

Fernandes, António Maximiano; Abdalhai, Mandour H; Ji, Jian; Xi, Bing-Wen; Xie, Jun; Sun, Jiadi; Noeline, Rasoamandrary; Lee, Byong H; Sun, Xiulan

2015-01-15

In this paper, we reported the construction of new high sensitive electrochemical genosensor based on multiwalled carbon nanotubes-chitosan-bismuth complex (MWCNT-Chi-Bi) and lead sulfide nanoparticles for the detection of pathogenic Aeromonas. Lead sulfide nanoparticles capped with 5'-(NH2) oligonucleotides thought amide bond was used as signalizing probe DNA (sz-DNA) and thiol-modified oligonucleotides sequence was used as fixing probe DNA (fDNA). The two probes hybridize with target Aeromonas DNA (tDNA) sequence (fDNA-tDNA-szDNA). The signal of hybridization is detected by differential pulse voltammetry (DPV) after electrodeposition of released lead nanoparticles (PbS) from sz-DNA on the surface of glass carbon electrode decorated with MWCNT-Chi-Bi, which improves the deposition and traducing electrical signal. The optimization of incubation time, hybridization temperature, deposition potential, deposition time and the specificity of the probes were investigated. Our results showed the highest sensibility to detect the target gene when compared with related biosensors and polymerase chain reaction (PCR). The detection limit for this biosensor was 1.0×10(-14) M. We could detect lower than 10(2) CFU mL(-1) of Aeromonas in spiked tap water. This method is rapid and sensitive for the detection of pathogenic bacteria and would become a potential application in biomedical diagnosis, food safety and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

Fishing in the Water: Effect of Sampled Water Volume on Environmental DNA-Based Detection of Macroinvertebrates.

PubMed

Mächler, Elvira; Deiner, Kristy; Spahn, Fabienne; Altermatt, Florian

2016-01-05

Accurate detection of organisms is crucial for the effective management of threatened and invasive species because false detections directly affect the implementation of management actions. The use of environmental DNA (eDNA) as a species detection tool is in a rapid development stage; however, concerns about accurate detections using eDNA have been raised. We evaluated the effect of sampled water volume (0.25 to 2 L) on the detection rate for three macroinvertebrate species. Additionally, we tested (depending on the sampled water volume) what amount of total extracted DNA should be screened to reduce uncertainty in detections. We found that all three species were detected in all volumes of water. Surprisingly, however, only one species had a positive relationship between an increased sample volume and an increase in the detection rate. We conclude that the optimal sample volume might depend on the species-habitat combination and should be tested for the system where management actions are warranted. Nevertheless, we minimally recommend sampling water volumes of 1 L and screening at least 14 μL of extracted eDNA for each sample to reduce uncertainty in detections when studying macroinvertebrates in rivers and using our molecular workflow.

Metal-organic frameworks for precise inclusion of single-stranded DNA and transfection in immune cells.

PubMed

Peng, Shuang; Bie, Binglin; Sun, Yangzesheng; Liu, Min; Cong, Hengjiang; Zhou, Wentao; Xia, Yucong; Tang, Heng; Deng, Hexiang; Zhou, Xiang

2018-04-03

Effective transfection of genetic molecules such as DNA usually relies on vectors that can reversibly uptake and release these molecules, and protect them from digestion by nuclease. Non-viral vectors meeting these requirements are rare due to the lack of specific interactions with DNA. Here, we design a series of four isoreticular metal-organic frameworks (Ni-IRMOF-74-II to -V) with progressively tuned pore size from 2.2 to 4.2 nm to precisely include single-stranded DNA (ssDNA, 11-53 nt), and to achieve reversible interaction between MOFs and ssDNA. The entire nucleic acid chain is completely confined inside the pores providing excellent protection, and the geometric distribution of the confined ssDNA is visualized by X-ray diffraction. Two MOFs in this series exhibit excellent transfection efficiency in mammalian immune cells, 92% in the primary mouse immune cells (CD4+ T cell) and 30% in human immune cells (THP-1 cell), unrivaled by the commercialized agents (Lipo and Neofect).

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

PubMed Central

Krug, A K; Enderle, D; Karlovich, C; Priewasser, T; Bentink, S; Spiel, A; Brinkmann, K; Emenegger, J; Grimm, D G; Castellanos-Rizaldos, E; Goldman, J W; Sequist, L V; Soria, J -C; Camidge, D R; Gadgeel, S M; Wakelee, H A; Raponi, M; Noerholm, M; Skog, J

2018-01-01

Abstract Background A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials NCT01526928 PMID:29216356

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.

PubMed

Kumar Khanna, Vinod

2007-01-01

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.

Organic cation transporter 3 contributes to norepinephrine uptake into perivascular adipose tissue

PubMed Central

Jackson, William F.; Burnett, Robert; Wilson, James N.; Thompson, Janice M.; Watts, Stephanie W.

2015-01-01

Perivascular adipose tissue (PVAT) reduces vasoconstriction to norepinephrine (NE). A mechanism by which PVAT could function to reduce vascular contraction is by decreasing the amount of NE to which the vessel is exposed. PVATs from male Sprague-Dawley rats were used to test the hypothesis that PVAT has a NE uptake mechanism. NE was detected by HPLC in mesenteric PVAT and isolated adipocytes. Uptake of NE (10 μM) in mesenteric PVAT was reduced by the NE transporter (NET) inhibitor nisoxetine (1 μM, 73.68 ± 7.62%, all values reported as percentages of vehicle), the 5-hydroxytryptamine transporter (SERT) inhibitor citalopram (100 nM) with the organic cation transporter 3 (OCT3) inhibitor corticosterone (100 μM, 56.18 ± 5.21%), and the NET inhibitor desipramine (10 μM) with corticosterone (100 μM, 61.18 ± 6.82%). Aortic PVAT NE uptake was reduced by corticosterone (100 μM, 53.01 ± 10.96%). Confocal imaging of mesenteric PVAT stained with 4-[4-(dimethylamino)-styrl]-N-methylpyridinium iodide (ASP+), a fluorescent substrate of cationic transporters, detected ASP+ uptake into adipocytes. ASP+ (2 μM) uptake was reduced by citalopram (100 nM, 66.68 ± 6.43%), corticosterone (100 μM, 43.49 ± 10.17%), nisoxetine (100 nM, 84.12 ± 4.24%), citalopram with corticosterone (100 nM and 100 μM, respectively, 35.75 ± 4.21%), and desipramine with corticosterone (10 and 100 μM, respectively, 50.47 ± 5.78%). NET protein was not detected in mesenteric PVAT adipocytes. Expression of Slc22a3 (OCT3 gene) mRNA and protein in PVAT adipocytes was detected by RT-PCR and immunocytochemistry, respectively. These end points support the presence of a transporter-mediated NE uptake system within PVAT with a potential mediator being OCT3. PMID:26432838

Organic cation transporter 3 contributes to norepinephrine uptake into perivascular adipose tissue.

PubMed

Ayala-Lopez, Nadia; Jackson, William F; Burnett, Robert; Wilson, James N; Thompson, Janice M; Watts, Stephanie W

2015-12-01

Perivascular adipose tissue (PVAT) reduces vasoconstriction to norepinephrine (NE). A mechanism by which PVAT could function to reduce vascular contraction is by decreasing the amount of NE to which the vessel is exposed. PVATs from male Sprague-Dawley rats were used to test the hypothesis that PVAT has a NE uptake mechanism. NE was detected by HPLC in mesenteric PVAT and isolated adipocytes. Uptake of NE (10 μM) in mesenteric PVAT was reduced by the NE transporter (NET) inhibitor nisoxetine (1 μM, 73.68 ± 7.62%, all values reported as percentages of vehicle), the 5-hydroxytryptamine transporter (SERT) inhibitor citalopram (100 nM) with the organic cation transporter 3 (OCT3) inhibitor corticosterone (100 μM, 56.18 ± 5.21%), and the NET inhibitor desipramine (10 μM) with corticosterone (100 μM, 61.18 ± 6.82%). Aortic PVAT NE uptake was reduced by corticosterone (100 μM, 53.01 ± 10.96%). Confocal imaging of mesenteric PVAT stained with 4-[4-(dimethylamino)-styrl]-N-methylpyridinium iodide (ASP(+)), a fluorescent substrate of cationic transporters, detected ASP(+) uptake into adipocytes. ASP(+) (2 μM) uptake was reduced by citalopram (100 nM, 66.68 ± 6.43%), corticosterone (100 μM, 43.49 ± 10.17%), nisoxetine (100 nM, 84.12 ± 4.24%), citalopram with corticosterone (100 nM and 100 μM, respectively, 35.75 ± 4.21%), and desipramine with corticosterone (10 and 100 μM, respectively, 50.47 ± 5.78%). NET protein was not detected in mesenteric PVAT adipocytes. Expression of Slc22a3 (OCT3 gene) mRNA and protein in PVAT adipocytes was detected by RT-PCR and immunocytochemistry, respectively. These end points support the presence of a transporter-mediated NE uptake system within PVAT with a potential mediator being OCT3. Copyright © 2015 the American Physiological Society.

Electrocatalysis in DNA Sensors

PubMed Central

Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

2014-01-01

Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

Electrocatalysis in DNA Sensors.

PubMed

Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

2014-12-14

Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology.

HIV-1-RNA in seminal plasma correlates with detection of HIV-1-DNA in semen cells, but not with CMV shedding, among MSM on successful antiretroviral regimens.

PubMed

Gantner, Pierre; Assoumou, Lambert; Leruez-Ville, Marianne; David, Ludivine; Suzan-Monti, Marie; Costagliola, Dominique; Rouzioux, Christine; Ghosn, Jade

2016-11-01

Intermittent seminal HIV-RNA detection can occur in MSM despite concomitant plasma virological control on combined ART (cART). We undertook the present study to determine if seminal HIV detection was associated with seminal cytomegalovirus (CMV) detection or detection of HIV-infected cells in semen. Longitudinal semen samples from HIV-1-infected MSM on successful cART enrolled in the EVARIST ANRS EP 49 study were analysed. We first conducted a case-control analysis (ratio 1 : 3) to assess HIV-DNA detection in semen cells in the 20 patients with detectable HIV-RNA in seminal plasma (cases) matched with 60 participants with undetectable HIV-RNA (controls) based on total HIV-DNA load in blood cells. Second, we measured CMV-DNA in all seminal plasma samples. HIV-1-DNA in semen cells was detected on at least one sample visit in 12/20 cases and 11/60 controls. Detection of HIV-RNA in seminal plasma was associated significantly with the detection of HIV-DNA in semen cells [OR, 7.6 (95% CI, 2.1-28.4); P = 0.002] when adjusted on total HIV-DNA in blood cells. CMV-DNA was detected in 107/273 seminal plasma samples with a median value of 3.62 log 10 copies/mL (IQR, 2.83-4.38), yielding a prevalence of 39.2%. Seminal CMV-DNA shedding [OR, 1.5 (95% CI, 0.6-3.6); P = 0.343] was not associated with the risk of detection of HIV-RNA in seminal plasma. The presence of HIV-DNA in semen cells was predictive of HIV-RNA detection, suggesting that viral particles arise through local HIV replication by infected semen cells. Despite virological control, compartmentalization of HIV in the genital tract might act in residual replication and transmission. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Integration of single-molecule detection with magnetic separation for multiplexed detection of DNA glycosylases.

PubMed

Li, Chen-Chen; Zhang, Yan; Tang, Bo; Zhang, Chun-Yang

2018-06-05

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

Potential of DNA barcoding for detecting quarantine fungi.

PubMed

Gao, Ruifang; Zhang, Guiming

2013-11-01

The detection of live quarantine pathogenic fungi plays an important role in guaranteeing regional biological safety. DNA barcoding, an emerging species identification technology, holds promise for the reliable, quick, and accurate detection of quarantine fungi. International standards for phytosanitary guidelines are urgently needed. The varieties of quarantine fungi listed for seven countries/regions, the currently applied detection methods, and the status of DNA barcoding for detecting quarantine fungi are summarized in this study. Two approaches have been proposed to apply DNA barcoding to fungal quarantine procedures: (i) to verify the reliability of known internal transcribed spacer (ITS)/cytochrome c oxidase subunit I (COI) data for use as barcodes, and (ii) to determine other barcodes for species that cannot be identified by ITS/COI. As a unique, standardizable, and universal species identification tool, DNA barcoding offers great potential for integrating detection methods used in various countries/regions and establishing international detection standards based on accepted DNA barcodes. Through international collaboration, interstate disputes can be eased and many problems related to routine quarantine detection methods can be solved for global trade.

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences.

PubMed

Wang, Xin; Lau, Choiwan; Kai, Masaaki; Lu, Jianzhong

2013-05-07

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

A novel technology for the detection, enrichment, and separation of trace amounts of target DNA based on amino-modified fluorescent magnetic composite nanoparticles.

PubMed

Wang, Guannan; Su, Xingguang

2010-06-01

A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.

Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system.

PubMed

Gao, Xue; Niu, Lu; Su, Xingguang

2012-01-01

This manuscript reports a method for the detection of double-stranded DNA, based on Mn:ZnSe d-dots and intercalating agent doxorubicin (DOX). DOX can quench the photoluminescence (PL) of Mn:ZnSe d-dots through photoinduced electron transfer process, after binding with Mn:ZnSe d-dots. The addition of DNA can result in the formation of the Mn:ZnSe d-dots-DOX-DNA ternary complexes, the fluorescence of the Mn:ZnSe d-dots-DOX complexes would be further quenched by the addition of DNA, thus allowing the detection of DNA. The formation mechanism of the Mn:ZnSe d-dots-DOX-DNA ternary complexes was studied in detail in this paper. Under optimal conditions, the quenched fluorescence intensity of Mn:ZnSe d-dots-DOX system are perfectly described by Stern-Volmer equation with the concentration of hsDNA ranging from 0.006 μg mL(-1) to 6.4 μg mL(-1). The detection limit (S/N = 3) for hsDNA is 0.5 ng mL(-1). The proposed method was successfully applied to the detection of DNA in synthetic samples and the results were satisfactory.

Optimised detection of mitochondrial DNA strand breaks.

PubMed

Hanna, Rebecca; Crowther, Jonathan M; Bulsara, Pallav A; Wang, Xuying; Moore, David J; Birch-Machin, Mark A

2018-05-04

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

PCR-based detection of a rare linear DNA in cell culture.

PubMed

Saveliev, Sergei V.

2002-11-11

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

PCR-based detection of a rare linear DNA in cell culture

PubMed Central

2002-01-01

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. PMID:12734566

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA

PubMed Central

Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

ABSTRACT Background Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. Materials and methods A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Results Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log10 copies/ml and 6.95 ± 1.08 log10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. Conclusion HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35. PMID:29264316

Detection of regional DNA methylation using DNA-graphene affinity interactions.

PubMed

Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

2017-01-15

We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Presence of intrahepatic (total and ccc) HBV DNA is not predictive of HBV recurrence after liver transplantation.

PubMed

Hussain, Munira; Soldevila-Pico, Consuelo; Emre, Sukru; Luketic, Velimir; Lok, Anna S F

2007-08-01

Previous studies reported that hepatitis B virus (HBV) deoxyribonucleic acid (DNA) can be detected in livers of patients who received transplants for hepatitis B despite the absence of serological markers of HBV recurrence. Quantification of HBV DNA was not performed and presence of covalently closed circular (ccc) DNA was not analyzed in most studies. We aimed to quantify total and ccc HBV DNA in explant liver and post-orthotopic liver transplantation (OLT) biopsies and to correlate the values with HBV recurrence post-OLT. Frozen liver tissue from 34 patients (9 with explant liver only, 9 with explant liver and post-OLT liver biopsies, and 16 with post-OLT biopsies only) in the National Institutes of Health HBV-OLT study was examined using real-time polymerase chain reaction (PCR). Among the 18 patients with explant liver, 7 were hepatitis B e antigen (HBeAg)-positive, 8 had detectable serum HBV DNA, and 10 received antiviral therapy prior to OLT. Total and ccc HBV DNA was detected in explant livers of 17 and 16 patients, respectively. Of the 10 patients who received antiviral therapy pre-OLT, serum HBV DNA was undetectable in 8 at transplantation but 7 had detectable total and ccc HBV DNA in their explant liver. Of the 25 patients with post-OLT biopsies, total HBV DNA was detected in 83% and ccc DNA in 17% of 47 biopsies, although only 2 patients had HBV recurrence. In conclusion, total and ccc HBV DNA could be detected in explant livers of most patients despite antiviral therapy pre-OLT. Total but not ccc HBV DNA could be detected in post-OLT liver biopsies of most patients despite undetectable serum HBV DNA and hepatitis B surface antigen (HBsAg). Our findings suggest that occult HBV reinfection occurs in most HBV patients after OLT and continued administration of appropriate prophylactic therapy is important in preventing overt HBV recurrence. Copyright (c) 2007 AASLD.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

PubMed

Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P

2017-09-01

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs.

PubMed

Chen, Yuqi; Song, Yanyan; Wu, Fan; Liu, Wenting; Fu, Boshi; Feng, Bingkun; Zhou, Xiang

2015-04-25

A conveniently amplified DNA AND logic gate platform was designed for the highly sensitive detection of low-abundance DNA fragment inputs based on strand displacement reaction and rolling circle amplification strategy. Compared with others, this system can detect miRNAs in biological samples. The success of this strategy demonstrates the potential of DNA logic gates in disease diagnosis.

ctDNA DLBCL Detection Lancet Oncology

Cancer.gov

Measurement of circulating tumor DNA in blood can be used to detect disease recurrence in patients with a curable form of cancer known as diffuse large B-cell lymphoma (DLBCL). In most patients, measurement of ctDNA enabled detection of microscopic diseas

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

NASA Astrophysics Data System (ADS)

Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

2006-01-01

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

Health status and potential uptake of transgenic DNA by Japanese quail fed diets containing genetically modified plant ingredients over 10 generations.

PubMed

Korwin-Kossakowska, A; Sartowska, K; Tomczyk, G; Prusak, B; Sender, G

2016-06-01

The hypothesis assumes that feed containing GMOs affects animal health and results in the transgene product accumulating in the body. Therefore, the objective of the study was to evaluate the impact of genetically modified (GM) ingredients used in poultry diets on aspects of bird health status and accumulation of transgenic DNA in eggs, breast muscle and internal organs. A total of 10 generations of Japanese quail were fed three types of diets: group A - containing GM soya (Roundup Ready) and non-GM maize, group B - containing GM maize (MON810) and non-GM soya, and group C - containing non-GM soya and maize. Bird performance traits were monitored throughout the trial. In 17-week-old animals of each generation, health examination took place on birds from each group including post-mortem necropsy and histological organ evaluation. For the purpose of transgenic DNA detection, samples of selected important tissues were taken. A molecular screening method of PCR amplification was used. The analysis of the sectional examination of birds used in the current experiment did not indicate the existence of the pathological changes caused by pathogens, nutritional factors or of environmental nature. The histopathological changes occurred in all three dietary groups and there were no statistically significant differences between the groups. There was no transgene amplification - neither CaMV35S promoter sequence nor nos terminator sequence, in the samples derived from breast muscle, selected tissues and germinal discs (eggs). According to the obtained results, it was concluded that there was no negative effect of the use of GM soya or maize with regard to bird health status or to the presence of transgenic DNA in the final consumable product.

Application of environmental DNA to detect an endangered marine skate species in the wild.

PubMed

Weltz, Kay; Lyle, Jeremy M; Ovenden, Jennifer; Morgan, Jessica A T; Moreno, David A; Semmens, Jayson M

2017-01-01

Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10-15 m) in Macquarie Harbour (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbour (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was determined for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% saturation) and lower DO (20% saturation) levels, revealing that the eDNA can be detected for 4 and 16 hours respectively, after which eDNA concentration drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concentrations, it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing.

Application of environmental DNA to detect an endangered marine skate species in the wild

PubMed Central

Morgan, Jessica A. T.; Moreno, David A.

2017-01-01

Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10–15 m) in Macquarie Harbour (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbour (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was determined for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% saturation) and lower DO (20% saturation) levels, revealing that the eDNA can be detected for 4 and 16 hours respectively, after which eDNA concentration drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concentrations, it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing. PMID:28591215

Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay.

PubMed

Wang, Cuini; Cheng, Yuanyuan; Liu, Biao; Wang, Yuanyuan; Gong, Weiming; Qian, Yihong; Guan, Zhifang; Lu, Haikong; Gu, Xin; Shi, Mei; Zhou, Pingyu

2018-05-09

The aim of this work was to investigate the application of the nested PCR assay for the detection of Treponema pallidum (TP) DNA from the blood of patients with different stages of syphilis. In this study, a nested PCR method targeting the Tpp47 and polA genes (Tpp47-Tp-PCR and polA-Tp-PCR) was developed to detect TP-DNA in whole blood samples collected from 262 patients with different stages of syphilis (84 primary syphilis, 97 secondary syphilis, and 81 latent syphilis patients). The PCR assay detected T. pallidum DNA in 53.6% and 62.9% of the patients with primary and secondary syphilis, respectively, which was much higher than the detection levels in patients with latent syphilis (7.4%) (both p < 0.001). For primary syphilis, a low RPR (0-16) was correlated with a higher detection rate of TP-DNA, whereas for secondary syphilis, the higher detection rate of blood TP-DNA was correlated with higher blood RPR titers (at or beyond 32). For latent syphilis, TP-DNA was only detectable by PCR in the early phase of the latent infection. Thus, blood RPR titers were correlated with the blood T. pallidum burden, but the correlations varied with primary and secondary syphilis. The results indicate that nested PCR is a sensitive method for detecting blood TP-DNA and is especially useful for detecting early syphilis including primary syphilis and secondary syphilis. The findings also suggest that the PCR assay may be used to complement other methods to enhance the diagnosis of syphilis.

Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource-limited settings by Loop-Mediated Isothermal Amplification assay.

PubMed

Akram, Arifa; Islam, S M Rashedul; Munshi, Saif Ullah; Tabassum, Shahina

2018-05-16

Transmission of Hepatitis B Virus (HBV) usually occurs due to the transfusion of blood or blood products from chronic HBV (CHB) or occult HBV infected (OBI) patients. Besides serological tests e.g. HBsAg and anti-HBc (total), detection of HBV-DNA is necessary for the diagnosis of OBI patients. Different nucleic acid tests (NATs) including real-time-Polymerase Chain Reaction (qPCR) are used for the detect HBV-DNA. The NATs are expensive and require technical expertise which are barriers to introducing them in resource-limited settings. This study was undertaken to evaluate the use of Loop-Mediated Isothermal Amplification (LAMP) assay as an alternative to qPCR for the detection of HBV-DNA in CHB and potential OBI patients in resource-limited settings. Following the published protocols with some modifications, a LAMP assay was developed for detection of HBV-DNA by either using a heat block followed by detection in an agarose gel or using a qPCR thermocycler. The LAMP assay was applied to supernatant prepared from heat treated serum collected from CHB and potential OBI patients. HBV viral load in serum was measured by qPCR using a single step HBV-DNA quantification kit. Among 200 samples tested, qPCR was capable to detect HBV-DNA in 25.5% of cases, whereas LAMP assay detected HBV-DNA in 43.5% cases. The qPCR was able to detect 11 (9.16%) potential OBI cases, whereas LAMP assay identified HBV-DNA in 43 (35.83%) cases. In addition to tests for HBsAg and/or anti-HBc (total), detection of HBV-DNA by LAMP assay may aid in preventing post-transfusion HBV infection in resource-limited settings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

F-18 choline PET does not detect increased metabolism in F-18 fluoroethyltyrosine-negative low-grade gliomas.

PubMed

Roelcke, Ulrich; Bruehlmeier, Matthias; Hefti, Martin; Hundsberger, Thomas; Nitzsche, Egbert U

2012-01-01

Positron emission tomography (PET) with radiolabeled amino acids provides information on biopsy target and chemotherapy response in patients with low-grade gliomas (LGG). In this article, we addressed whether PET with F-18 choline (CHO) detects increased metabolism in F-18 fluoroethyltyrosine (FET)-negative LGG patients. Six LGG patients with nongadolinium-enhancing (magnetic resonance) FET-negative LGG were imaged with CHO PET. Regions of interest were positioned over tumor and contralateral brain. Uptake of FET and CHO was quantified as count ratio of tumor to contralateral brain. The mean FET uptake ratio for FET-negative LGG was 0.95 ± 0.03 (mean ± standard deviation). Five tumors did not show increased uptake ratios for CHO (0.96 ± 0.12). Slightly increased CHO uptake was found in 1 patient (1.24), which, however, was not associated with tumor visualization. Amino acid and choline uptake appear to behave similar in nongadolinium-enhancing LGG. For clinical purposes, CHO PET is not superior to FET PET.

Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum)

USGS Publications Warehouse

Goldberg, Caren S.; Sepulveda, Adam; Ray, Andrew; Baumgardt, Jeremy A.; Waits, Lisette P.

2013-01-01

Early detection of aquatic invasive species is a critical task for management of aquatic ecosystems. This task is hindered by the difficulty and cost of surveying aquatic systems thoroughly. The New Zealand mudsnail (Potamopyrgus antipodarum) is a small, invasive parthenogenic mollusk that can reach very high population densities and severely affects ecosystem functioning. To assist in the early detection of this invasive species, we developed and validated a highly sensitive environmental deoxyribonucleic acid (eDNA) assay. We used a dose–response laboratory experiment to investigate the relationship between New Zealand mudsnail density and eDNA detected through time. We documented that as few as 1 individual in 1.5 L of water for 2 d could be detected with this method, and that eDNA from this species may remain detectable for 21 to 44 d after mudsnail removal. We used the eDNA method to confirm the presence of New Zealand mudsnail eDNA at densities as low as 11 to 144 snails/m2 in a eutrophic 5th-order river. Combined, these results demonstrate the high potential for eDNA surveys to assist with early detection of a widely distributed invasive aquatic invertebrate.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

PubMed

Han, Daehoon; Hong, Jinkee; Kim, Hyun Cheol; Sung, Jong Hwan; Lee, Jong Bum

2013-11-01

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

PubMed Central

Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

2016-01-01

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

[Increased glucose uptake by seborrheic keratosis on PET scan].

PubMed

Merklen-Djafri, C; Truntzer, P; Hassler, S; Cribier, B

2017-05-01

Positron emission tomography (PET) is an examination based upon the uptake of a radioactive tracer by hypermetabolic cells. It is primarily used in tandem with tomodensitometry (PET-TDM) for cancer staging because of its high sensitivity and specificity for the detection of metastases. However, unusually high uptake may occur with benign tumours, including skin tumours. Herein, we report an extremely rare case of pathological uptake levels resulting from seborrhoeic keratosis. A 55-year-old male patient with oesophageal squamous-cell carcinoma was referred to us following the discovery of an area of high marker uptake following PET-TDM and corresponding to a pigmented skin lesion. No other areas of suspect high uptake were seen. The lesion was surgically excised and histological examination indicated seborrhoeic keratosis. The histological appearance was that of standard seborrhoeic keratosis without any notable mitotic activity. PET-TDM is an examination that enables diagnosis of malignancy. However, rare cases have been described of increased marker uptake by benign cutaneous tumours such as histiocytofibroma, pilomatricoma and condyloma. To date, there have only been only very few cases of increased uptake due to seborrhoeic keratosis. This extremely unusual case of increased glucose uptake in PET-TDM due to seborrhoeic keratosis confirms that the hypermetabolic activity detected by this examination is not necessarily synonymous with malignancy and that confirmation by clinical and histological findings is essential. The reasons for increased metabolic activity within such benign tumours are not known. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Youyu; Tang, Zhiwen; Wang, Jun

2010-08-01

A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate duemore » to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.« less

Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

PubMed

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2007-06-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E. coli O157:H7 DNA detection.

PubMed

Wang, Lijiang; Liu, Qingjun; Hu, Zhaoying; Zhang, Yuanfan; Wu, Chunsheng; Yang, Mo; Wang, Ping

2009-05-15

A novel biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for Escherichia coli O157:H7 DNA detection. An original and dynamic polymerase-extending (PE) DNA hybridization procedure is proposed, where hybridization happens in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strand would be extended as long as the target DNA strand, then the capability to block the ionic flow in the pores has been prominently enhanced by the double strand complex. We have investigated the variation of ionic conductivity during the fabrication of the film and the hybridization using cyclic voltammetry and impedance spectroscopy. The present approach provides low detection limit for DNA (a few hundreds of pmol), rapid label-free and easy-to-use bacteria detection, which holds the potential for future use in various ss-DNA analyses by integrated into a self-contained biochip.

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

PubMed

Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

2009-02-02

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Monitoring carnosine uptake by RAW 264.7 macrophage cells using microchip electrophoresis with fluorescence detection

PubMed Central

Fresta, Claudia G.; Hogard, Michael L.; Caruso, Giuseppe; Melo Costa, Elton E.; Lazzarino, Giuseppe; Lunte, Susan M.

2017-01-01

Carnosine, a dipeptide found in a variety of tissues, is believed to possess antioxidant properties. It serves as a scavenger of reactive nitrogen and oxygen species (RNOS), which are important stress mediators of pro-inflammatory conditions and can lead to macrophage activation. In this study, intracellular concentrations of carnosine in murine RAW 264.7 macrophage cells were determined using microchip electrophoresis with laser-induced fluorescence detection following derivatization with naphthalene-2,3-dicarboxaldehyde and cyanide. The method was linear from 25 nM to 5 μM with a limit of detection in cell lysate samples of 65 nM. Using the method of standard additions, the basal intracellular content of carnosine in macrophage cells was determined to be 0.079 ± 0.02 nmol/106 cells. The uptake of carnosine by these cells was then investigated under both physiological and pro-inflammatory conditions. There was a 2.8-fold increase in carnosine uptake for macrophages exposed to lipopolysaccharide and interferon-γ prior to incubation, compared to the controls. This suggests that macrophages may use carnosine uptake as a defense mechanism under pro-inflammatory conditions. Future studies will investigate the role of the carnosine transporter in carnosine uptake and its possible correlation with cell morphological changes observed after stimulation. PMID:29104617

Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

PubMed Central

Gnana-Prakasam, Jaya P.; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Reddy, Sushma K.; Martin, Pamela M.; Thangaraju, Muthusamy; Smith, Sylvia B.; Ganapathy, Vadivel

2013-01-01

Purpose. Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Methods. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Results. Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells. Conclusions. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells. PMID:23169885

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

PubMed

Mazurek, Agnieszka M; Fiszer-Kierzkowska, A; Rutkowski, T; Składowski, K; Pierzyna, M; Scieglińska, D; Woźniak, G; Głowacki, G; Kawczyński, R; Małusecka, E

2013-01-01

The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.

A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

PubMed

Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

2016-12-15

DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection.

PubMed

Ma, Long; Sun, Nana; Zhang, Jinyan; Tu, Chunhao; Cao, Xiuqi; Duan, Demin; Diao, Aipo; Man, Shuli

2017-11-23

We report a novel assembly of polyethyleneimine (PEI)-coated Fe 3 O 4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein-DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.

High Sensitivity Detection of CdSe/ZnS Quantum Dot-Labeled DNA Based on N-type Porous Silicon Microcavities

PubMed Central

Lv, Changwu; Jia, Zhenhong; Lv, Jie; Zhang, Hongyan; Li, Yanyu

2017-01-01

N-type macroporous silicon microcavity structures were prepared using electrochemical etching in an HF solution in the absence of light and oxidants. The CdSe/ZnS water-soluble quantum dot-labeled DNA target molecules were detected by monitoring the microcavity reflectance spectrum, which was characterized by the reflectance spectrum defect state position shift resulting from changes to the structures’ refractive index. Quantum dots with a high refractive index and DNA coupling can improve the detection sensitivity by amplifying the optical response signals of the target DNA. The experimental results show that DNA combined with a quantum dot can improve the sensitivity of DNA detection by more than five times. PMID:28045442

High Sensitivity Detection of CdSe/ZnS Quantum Dot-Labeled DNA Based on N-type Porous Silicon Microcavities.

PubMed

Lv, Changwu; Jia, Zhenhong; Lv, Jie; Zhang, Hongyan; Li, Yanyu

2017-01-01

N-type macroporous silicon microcavity structures were prepared using electrochemical etching in an HF solution in the absence of light and oxidants. The CdSe/ZnS water-soluble quantum dot-labeled DNA target molecules were detected by monitoring the microcavity reflectance spectrum, which was characterized by the reflectance spectrum defect state position shift resulting from changes to the structures' refractive index. Quantum dots with a high refractive index and DNA coupling can improve the detection sensitivity by amplifying the optical response signals of the target DNA. The experimental results show that DNA combined with a quantum dot can improve the sensitivity of DNA detection by more than five times.

Detection and quantification of 223Ra uptake in bone metastases of patients with castration resistant prostate carcinoma, with the aim of determining the absorbed dose in the metastases.

PubMed

Mínguez, P; Gómez de Iturriaga, A; Fernández, I L; Rodeño, E

To obtain the necessary acquisition and calibration parameters in order to evaluate the possibility of detecting and quantifying 223 Ra uptake in bone metastases of patients treated for castration resistant prostate carcinoma. Furthermore, in the cases in which the activity can be quantified, to determine the absorbed dose. Acquisitions from a Petri dish filled with 223 Ra were performed in the gamma camera. Monte Carlo simulations were also performed to study the partial volume effect. Formulae to obtain the detection and quantification limits of 223 Ra uptake were applied to planar images of two patients 7 days post-administration of 55kBq/kg of 223 Ra. In order to locate the lesions in advance, whole-body scans and SPECT/CT images were acquired after injecting 99m Tc-HDP. The optimal energy window was found to be at 82keV with a medium-energy collimator MEGP. Of the lesions found in the patients, only those that had been detected in both the AP and PA projections could be quantified. These lesions were those which had shown a higher 99m Tc-HDP uptake. The estimated values of absorbed doses ranged between 0.7Gy and 7.8Gy. Of the lesions that can be detected, it is not possible to quantify the activity uptake in some of them, which means that the absorbed dose cannot be determined either. This does not mean that the absorbed dose in these lesions can be regarded as negligible. Copyright © 2017 Elsevier España, S.L.U. y SEMNIM. All rights reserved.

Report: An ex vivo up-take of levamisole molecules by cestode (Monezia expensa) of goat (Capra hirsa) and its detection through RP-HPLC.

PubMed

Ayaz, Muhammad Mazhar; Sajid, Muhammad; Das, Sanjota Nirmal; Hanif, Muhammad

2018-05-01

Detection of various molecules of drugs remained a prime issue especially in tissues of animals, humans and in their target parasites. The cestode/tapeworms pose a dilemma because of their weird body composition and uptake pattern of nutrients and medicines especially through absorption by tegument. We selected levamisole; thought to be potent antiparasitic/ani-cestodal drug. The uptake of levamisole (LEV) through cestodeal tissues is studied through HPCL in this paper. High performance liquid chromatography technique has been utilized to know the uptake of levamisole in tissues of cestodes of Goat (Monezia expensa) in small ruminants. The drug was exposed to M. expensa by in vitro till its death or a parasite ceases its movement. The tissue/ part of proglattids of the M. expensa were homogenized with some modifications and levamisole extraction was performed with liquid phase extraction method. The evaporation of solvent was done and the residual cestodal tissues were cleaned by solid phase. After the solid phase extraction method, the recovery of drug, detection and quantification of levamisole from cestodal tissues was determined through Reverse Phase Column High Performance Liquid Chromatography (RP-HPLC). Levamisole (LEV) molecules assay was obtained on a C18 reverse-phase (20um, 6mm x 150mm) column at flow rate of 1ml/min using acetonitrile and ammonium acetate as mobile phase and UV detection was done at 254nm. The development of method of Levamisole (LEV) detection from cestodal tissues by HPLC in vitro samples has been demonstrated first time in Pakistan, which can provide the solution of parasitic control and provide in sight in to the uptake of anti cestodal drugs either against human or livestock parasites.

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier

PubMed Central

Baghirov, Habib; Karaman, Didem; Viitala, Tapani; Duchanoy, Alain; Lou, Yan-Ru; Mamaeva, Veronika; Pryazhnikov, Evgeny; Khiroug, Leonard; de Lange Davies, Catharina; Sahlgren, Cecilia; Rosenholm, Jessica M.

2016-01-01

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport. PMID:27547955

Detection of an enigmatic plethodontid Salamander using Environmental DNA

USGS Publications Warehouse

Pierson, Todd W.; Mckee, Anna; Spear, Stephen F.; Maerz, John C.; Camp, Carlos D.; Glenn, Travis C.

2016-01-01

The isolation and identification of environmental DNA (eDNA) offers a non-invasive and efficient method for the detection of rare and secretive aquatic wildlife, and it is being widely integrated into inventory and monitoring efforts. The Patch-Nosed Salamander (Urspelerpes brucei) is a tiny, recently discovered species of plethodontid salamander known only from headwater streams in a small region of Georgia and South Carolina. Here, we present results of a quantitative PCR-based eDNA assay capable of detecting Urspelerpes in more than 75% of 33 samples from five confirmed streams. We deployed the method at 31 additional streams and located three previously undocumented populations of Urspelerpes. We compare the results of our eDNA assay with our attempt to use aquatic leaf litterbags for the rapid detection of Urspelerpes and demonstrate the relative efficacy of the eDNA assay. We suggest that eDNA offers great potential for use in detecting other aquatic and semi-aquatic plethodontid salamanders.

Detection and persistence of environmental DNA from an invasive, terrestrial mammal.

PubMed

Williams, Kelly E; Huyvaert, Kathryn P; Vercauteren, Kurt C; Davis, Amy J; Piaggio, Antoinette J

2018-01-01

Invasive Sus scrofa , a species commonly referred to as wild pig or feral swine, is a destructive invasive species with a rapidly expanding distribution across the United States. We used artificial wallows and small waterers to determine the minimum amount of time needed for pig eDNA to accumulate in the water source to a detectable level. We removed water from the artificial wallows and tested eDNA detection over the course of 2 weeks to understand eDNA persistence. We show that our method is sensitive enough to detect very low quantities of eDNA shed by a terrestrial mammal that has limited interaction with water. Our experiments suggest that the number of individuals shedding into a water system can affect persistence of eDNA. Use of an eDNA detection technique can benefit management efforts by providing a sensitive method for finding even small numbers of individuals that may be elusive using other methods.

DNA-magnetic bead detection using disposable cards and the anisotropic magnetoresistive sensor

NASA Astrophysics Data System (ADS)

Hien, L. T.; Quynh, L. K.; Huyen, V. T.; Tu, B. D.; Hien, N. T.; Phuong, D. M.; Nhung, P. H.; Giang, D. T. H.; Duc, N. H.

2016-12-01

A disposable card incorporating specific DNA probes targeting the 16 S rRNA gene of Streptococcus suis was developed for magnetically labeled target DNA detection. A single-stranded target DNA was hybridized with the DNA probe on the SPA/APTES/PDMS/Si as-prepared card, which was subsequently magnetically labeled with superparamagnetic beads for detection using an anisotropic magnetoresistive (AMR) sensor. An almost linear response between the output signal of the AMR sensor and amount of single-stranded target DNA varied from 4.5 to 18 pmol was identified. From the sensor output signal response towards the mass of magnetic beads which were directly immobilized on the disposable card surface, the limit of detection was estimated about 312 ng ferrites, which corresponds to 3.8 μemu. In comparison with DNA detection by conventional biosensor based on magnetic bead labeling, disposable cards are featured with higher efficiency and performances, ease of use and less running cost with respects to consumables for biosensor in biomedical analysis systems operating with immobilized bioreceptor.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

PubMed

Belmonte, Frances R; Martin, James L; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A; Kaufman, Brett A

2016-04-28

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

PubMed Central

Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

2016-01-01

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

Evaluation of basal DNA damage and oxidative stress in Wistar rat leukocytes after exposure to microwave radiation.

PubMed

Garaj-Vrhovac, Vera; Gajski, Goran; Trosić, Ivancica; Pavicić, Ivan

2009-05-17

The aim of this study was to assess whether microwave-induced DNA damage is basal or it is also generated through reactive oxygen species (ROS) formation. After having irradiated Wistar rats with 915MHz microwave radiation, we assessed different DNA alterations in peripheral leukocytes using standard and formamidopyrimidine DNA-glycosylase (Fpg)-modified comet assay. The first is a sensitive tool for detecting primary DNA damage, and the second is much more specific for detecting oxidative damage. The animals were irradiated for 1h a day for 2 weeks at a field power density of 2.4W/m(2), and the whole-body average specific absorption rate (SAR) of 0.6W/kg. Both the standard and the Fpg-modified comet assay detected increased DNA damage in blood leukocytes of the exposed rats. The significant increase in Fpg-detected DNA damage in the exposed rats suggests that oxidative stress is likely to be responsible. DNA damage detected by the standard comet assay indicates that some other mechanisms may also be involved. In addition, both methods served proved sensitive enough to measure basal and oxidative DNA damage after long-term exposure to 915MHz microwave radiation in vivo.

Status and prospects of DNA barcoding in medically important parasites and vectors.

PubMed

Ondrejicka, Danielle A; Locke, Sean A; Morey, Kevin; Borisenko, Alex V; Hanner, Robert H

2014-12-01

For over 10 years, DNA barcoding has been used to identify specimens and discern species. Its potential benefits in parasitology were recognized early, but its utility and uptake remain unclear. Here we review studies using DNA barcoding in parasites and vectors affecting humans and find that the technique is accurate (accords with author identifications based on morphology or other markers) in 94-95% of cases, although aspects of DNA barcoding (vouchering, marker implicated) have often been misunderstood. In a newly compiled checklist of parasites, vectors, and hazards, barcodes are available for 43% of all 1403 species and for more than half of 429 species of greater medical importance. This is encouraging coverage that would improve with an active campaign targeting parasites and vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection

NASA Astrophysics Data System (ADS)

Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

2014-09-01

The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03795b

An eDNA assay for river otter detection: A tool for surveying a semi-aquatic mammal

Treesearch

Ticha M. Padgett-Stewart; Taylor M. Wilcox; Kellie J. Carim; Kevin S. McKelvey; Michael K. Young; Michael K. Schwartz

2016-01-01

Environmental DNA (eDNA) is an effective tool for the detection of elusive or low-density aquatic organisms. However, it has infrequently been applied to mammalian species. North American river otters (Lontra canadensis) are both broad ranging and semi-aquatic, making them an ideal candidate for examining the uses of eDNA for detection of mammals. We developed...

Direct Detection and Sequencing of Damaged DNA Bases

PubMed Central

2011-01-01

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

Direct detection and sequencing of damaged DNA bases.

PubMed

Clark, Tyson A; Spittle, Kristi E; Turner, Stephen W; Korlach, Jonas

2011-12-20

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

DNA hybridization sensor based on pentacene thin film transistor.

PubMed

Kim, Jung-Min; Jha, Sandeep Kumar; Chand, Rohit; Lee, Dong-Hoon; Kim, Yong-Sang

2011-01-15

A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs. Copyright © 2010 Elsevier B.V. All rights reserved.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

PubMed

Sawamura, Kensuke; Hashimoto, Masahiko

2017-01-01

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

Somatostatin receptor scintigraphy in patients with rheumatoid arthritis and secondary Sjögren's syndrome treated with Infliximab: a pilot study.

PubMed

Anzola-Fuentes, L K; Chianelli, M; Galli, F; Glaudemans, A W J M; Martin Martin, L; Todino, V; Migliore, A; Signore, A

2016-12-01

Human T lymphocytes infiltrating tissues in autoimmune diseases are known to express somatostatin receptors amongst other activation markers. In this study, we evaluated whether somatostatin receptor scintigraphy (SRS) using a radiolabelled somatostatin analogue ((99m)Tc-EDDA/tricine-HYNIC-tyr(3)-octreotide ((99m)Tc-EDDA/HYNIC-TOC)) is able to detect the presence of immune-mediated processes in patients with rheumatoid arthritis and secondary Sjögren's syndrome. We also aimed to evaluate whether positivity to SRS was predictive of therapeutic response and if SRS could be used for monitoring the efficacy of immunomodulatory treatment. Eighteen patients with rheumatoid arthritis and secondary Sjögren's syndrome not responding to conventional treatment were recruited for treatment with infliximab, a monoclonal antibody against TNF-α. All patients had complete blood cell count, renal and liver function tests, measurements of ESR, CRP, ANA, ENA, and anti-dsDNA antibodies, functional salivary gland scintigraphy, labial biopsy, and ophthalmologic assessment with Schirmer's test and tear film break-up time (BUT). Diagnosis was made according to the revised criteria of the American-European Consensus Group. All patients underwent SRS at baseline and after 3-6 months of therapy with infliximab. Eleven out of 18 had repeat SRS images. Images of the salivary glands and major joints were acquired 3 h after injection of 370 MBq of (99m)Tc-EDDA/HYNIC-TOC. Image analysis was performed semi-quantitatively. All patients showed uptake of (99m)Tc-EDDA/HYNIC-TOC in the joints. Salivary glands also showed variable radiopharmaceutical uptake in 12 out of 18 patients, but all patients showed presence of lymphocytic infiltration at labial salivary gland biopsy. All patients, who repeated the study after treatment, showed significant reduction of somatostatin uptake in the joints but not in the salivary glands. SRS using (99m)Tc-EDDA/HYNIC-TOC may be a useful imaging tool to assess disease activity and extent in patients with rheumatoid arthritis and may help to detect secondary Sjögren's syndrome. It may also aid therapy decision-making with anti-TNFα antibodies in the joints but not in salivary glands.

Ga-68 DOTA-NOC uptake in the pancreas: pathological and physiological patterns.

PubMed

Krausz, Yodphat; Rubinstein, Rina; Appelbaum, Liat; Mishani, Eyal; Orevi, Marina; Fraenkel, Merav; Tshori, Sagi; Glaser, Benjamin; Bocher, Moshe; Salmon, Asher; Chisin, Roland; Gross, David J; Freedman, Nanette

2012-01-01

Gallium-68 (Ga-68) DOTA-1-NaI3-octreotide (DOTA-NOC) positron emission tomography (PET)/computed tomography (CT) is increasingly used for neuroendocrine tumors (NETs), often found primarily in the pancreas. However, physiologic uptake of DOTA-NOC has been described in the uncinate process of the pancreas. We studied DOTA-NOC uptake in this organ. Ninety-six patients underwent 103 DOTA-NOC scans, with pathology-proven pancreatic NET (n = 40) and nonpancreatic NET or biochemical suspicion of NET (n = 63). DOTA-NOC uptake was detected in 35 documented pancreatic tumor sites (SUV: 5.5-165; mean: 25.7 ± 28.8; median: 17.8). Among 63 cases without previous known pathology, uptake was suspicious for tumor in 24 sites (SUV: 4.7-35; mean 16.3 ± 8.0; median: 14.1), and in 38 sites, it was judged as physiological, generally lower relative to adjacent structures (SUV: 2.2-12.6; mean: 6.6 ± 2.2; median: 6.2). In 24 scans with suspected tumor and in 37 of 38 scans with physiological uptake, diagnostic computed tomography or magnetic resonance imaging or endoscopic ultrasonography failed to detect tumor. Pancreatic DOTA-NOC uptake must be interpreted with caution, and further studies are required.

Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

PubMed

Saw, Constance Lay Lay; Olivo, Malini; Wohland, Thorsten; Fu, Chit Yaw; Kho, Kiang Wei; Soo, Khee Chee; Sia Heng, Paul Wan

2007-10-01

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, M.V., E-mail: evans.marina@epa.go; Caldwell, J.C.

Dichloromethane (DCM, methylene chloride) is a lipophilic volatile compound readily absorbed and then metabolized to several metabolites that may lead to chronic toxicity in different target organs. Physiologically based pharmacokinetic (PBPK) models are useful tools for calculation of internal and target organ doses of parent compound and metabolites. PBPK models, coupled with in vivo inhalation gas-uptake data, can be useful to estimate total metabolism. Previously, such an approach was used to make predictions regarding the metabolism and to make subsequent inferences of DCM's mode of action for toxicity. However, current evidence warrants re-examination of this approach. The goal of thismore » work was to examine two different hypotheses for DCM metabolism in mice. One hypothesis describes two metabolic pathways: one involving cytochrome P450 2E1 (CYP2E1) and a second glutathione (GSH). The second metabolic hypothesis describes only one pathway mediated by CYP2E1 that includes multiple binding sites. The results of our analysis show that the in vivo gas-uptake data fit both hypotheses well and the traditional analysis of the chamber concentration data is not sufficient to distinguish between them. Gas-uptake data were re-analyzed by construction of a velocity plot as a function of increasing DCM initial concentration. The velocity (slope) analysis revealed that there are two substantially different phases in velocity, one rate for lower exposures and a different rate for higher exposures. The concept of a 'metabolic switch,' namely that due to conformational changes in the enzyme after one site is occupied - a different metabolic rate is seen - is also consistent with the experimental data. Our analyses raise questions concerning the importance of GSH metabolism for DCM. Recent research results also question the importance of this pathway in the toxicity of DCM. GSH-related DNA adducts were not formed after in vivo DCM exposure in mice and DCM-induced DNA damage has been detected in human lung cultures without GSH metabolism. In summary, a revised/updated metabolic hypothesis for DCM has been examined using in vivo inhalation data in mice combined with PBPK modeling that is consistent with up-to-date models of the active site for CYP2E1 and suggests that this pathway is the major metabolizing pathway for DCM metabolism.« less

Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+-dependent DNAzyme cleavage.

PubMed

Yin, Huan-Shun; Li, Bing-Chen; Zhou, Yun-Lei; Wang, Hai-Yan; Wang, Ming-Hui; Ai, Shi-Yun

2017-10-15

MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg 2+ -dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg 2+ , trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma.

PubMed

Lim, Ji Hyae; Kim, Mee Jin; Kim, Shin Young; Kim, Hye Ok; Song, Mee Jin; Kim, Min Hyoung; Park, So Yeon; Yang, Jae Hyug; Ryu, Hyun Mee

2011-02-01

To perform a reliable non-invasive detection of the fetal achondroplasia using maternal plasma. We developed a quantitative fluorescent-polymerase chain reaction (QF-PCR) method suitable for detection of the FGFR3 mutation (G1138A) causing achondroplasia. This method was applied in a non-invasive detection of the fetal achondroplasia using circulating fetal-DNA (cf-DNA) in maternal plasma. Maternal plasmas were obtained at 27 weeks of gestational age from women carrying an achondroplasia fetus or a normal fetus. Two percent or less achondroplasia DNA was reliably detected by QF-PCR. In a woman carrying a normal fetus, analysis of cf-DNA showed only one peak of the wild-type G allele. In a woman expected an achondroplasia fetus, analysis of cf-DNA showed the two peaks of wild-type G allele and mutant-type A allele and accurately detected the fetal achondroplasia. The non-invasive method using maternal plasma and QF-PCR may be useful for diagnosis of the fetal achondroplasia.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

PubMed

Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

2016-05-01

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

The DNA isolation method has effect on allele drop-out and on the results of fluorescent PCR and DNA fragment analysis.

PubMed

Nagy, Bálint; Bán, Zoltán; Papp, Zoltán

2005-10-01

The quality and the quantity of isolated DNA have an effect on PCR amplifications. The authors studied three DNA isolation protocols (resin binding method using fresh and frozen amniotic fluid samples, and silica adsorption method using fresh samples) on the quantity and on the quality of the isolated DNA. Amniotic fluid samples were obtained from 20 pregnant women. The isolated DNA concentrations were determined by real-time fluorimeter using SYBRGreen I method. Each sample was studied for the presence of 8 STR markers. The authors compared the number of the detected alleles, electrophoretograms and peak areas. There was a significant difference between the concentration of the obtained DNA and in the peak areas between the three isolation protocols. The numbers of detected alleles were different, we observed the most allele drop outs in the resin type DNA isolation protocol from the fresh sample (detected allele numbers 182), followed by resin binding protocol from the frozen samples (detected allele number 243) and by the silica adsorption method (detected allele number 264). The authors demonstrated that the DNA isolation method has an effect on the quantity and quality of the isolated DNA, and on further PCR amplifications.

The Chemical and Biological Effects of cis-Dichlorodiammineplatinum (II), an Antitumor Agent, on DNA

PubMed Central

Munchausen, Linda L.

1974-01-01

cis-Dichlorodiammineplatinum (II) binds irreversibly to the bases in DNA; the amount of platinum complex bound can be determined from changes in the ultraviolet absorption spectrum. As the ratio of platinum to phosphate is increased, an increasing inactivation of bacterial transforming DNA is observed. At a ratio that corresponds to spectrometric saturation, transforming activity is inactivated >105-fold. The trans isomer of the platinum complex, which is not effective against tumors, induces a similar inactivation of transforming DNA but with half the efficiency, indicating a different mode of binding. The sensitivity to inactivation by cis isomer varies slightly with the genetic marker assayed but is not dependent on the excision repair system. Uptake of DNA by competent cells is unaffected by bound platinum complex; however, integration of platinum-bound transforming DNA into the host genome decreases as the mole fraction of platinum increases. This loss of integration parallels the decreased transforming activity of the DNA. Although the drug induces interstrand crosslinks in DNA in vitro, these crosslinks are relatively rare events and cannot account for the observed inactivation. PMID:4548188

Direct Imaging of Gene-Carrier Complexes in Animal Cells

NASA Astrophysics Data System (ADS)

Lin, Alison J.; Slack, Nelle L.; Ahmad, Ayesha; Matsumoto, Brian; Safinya, Cyrus R.

1998-03-01

Cationic lipids are promising gene carriers for DNA transfection. Establishing the correlations between structures of cationic lipid/DNA complexes (CL-DNA) and pathways of transfection will greatly aid us in achieving the optimal CL-DNA transfections. Our first step is to determine the uptake mechanism of DNA by studying the interactions and structures of DNA and cationic lipids. X-ray diffraction shows that the CL-DNA undergoes structural phase transitions from lamellar( J. Raedler, I. Koltover, T. Salditt, C. R. Safinya, Science 275, 810 (1997).) to inverted hexagonal self-assemblies as we change the lipid composition. X-ray diffraction and optical microscopy techniques are used to directly image the progress of the CL-DNA in mouse L-cells and unravel the complex structure in-situ. Fluorescence and confocal optical microscopy techniques allow us to monitor the interactions between the complexes and different organelles in the cell cytoplasm. Current results indicate that once inside cells, complexes containing DOPE follow a different pathway from those containing DOPC. This research is funded by NSF-DMR-9624091, PRF-31352-AC7, and Los Alamos-STB/UC:96-108.

Expression and characterization of a β-fructofuranosidase from the parasitic protist Trichomonas vaginalis

PubMed Central

2014-01-01

Background Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present. Results We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar Km and Vmax to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin. Conclusions T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose. PMID:24972630

Expression and characterization of a β-fructofuranosidase from the parasitic protist Trichomonas vaginalis.

PubMed

Dirkx, Michael; Boyer, Michael P; Pradhan, Prajakta; Brittingham, Andrew; Wilson, Wayne A

2014-06-28

Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present. We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar K(m) and V(max) to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin. T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

PubMed Central

Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2013-01-01

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

PubMed Central

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

Potentiometric Aptasensing of Vibrio alginolyticus Based on DNA Nanostructure-Modified Magnetic Beads.

PubMed

Zhao, Guangtao; Ding, Jiawang; Yu, Han; Yin, Tanji; Qin, Wei

2016-12-02

A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10-100 CFU mL -1 with a detection limit of 10 CFU mL -1 , and a good specificity for the detection of Vibrio alginolyticus . This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures.

[Detection of Toxoplasma gondii DNA in human lymph node tissue by in situ hybridization].

PubMed

Liu, C; Ke, O; Tan, D; Zhang, Z

1998-01-01

To detect the presence of Toxoplasma gondii in lymph node tissue in patients with Toxoplasma infection. T. gondii (RH strain) specific DNA fragment clones were obtained by using PCR and gene recombination technique. The DNA fragments used as hybridization probes were labelled with digoxigenin by random primer method. The technique of in situ hybridization (ISH) was used to detect T. g DNA in the lymph node sections. Four out of 120 samples T. g DNA were found positive, one with Hodgkin's disease (HD) (1/32), one with non-Hodgkin's lymphoma (NHL) (1/41) and 2 with chronic lymphadenitis (CL) (2/47). The total positive rate was 3.3%. It was demonstrated that this highly specific probe could detect 10 pg of the total RH strain T. g DNA. ISH was applicable in detecting pathogens in the lymph node tissues of individuals with Toxoplasma infection.

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

Interaction of dacarbazine and imexon, in vitro and in vivo, in human A375 melanoma cells.

PubMed

Samulitis, Betty K; Dorr, Robert T; Chow, H-H Sherry

2011-09-01

We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.

Transferrin-bearing polypropylenimine dendrimer for targeted gene delivery to the brain.

PubMed

Somani, Sukrut; Blatchford, David R; Millington, Owain; Stevenson, M Lynn; Dufès, Christine

2014-08-28

The possibility of using genes as medicines to treat brain diseases is currently limited by the lack of safe and efficacious delivery systems able to cross the blood-brain barrier, thus resulting in a failure to reach the brain after intravenous administration. On the basis that iron can effectively reach the brain by using transferrin receptors for crossing the blood-brain barrier, we propose to investigate if a transferrin-bearing generation 3-polypropylenimine dendrimer would allow the transport of plasmid DNA to the brain after intravenous administration. In vitro, the conjugation of transferrin to the polypropylenimine dendrimer increased the DNA uptake by bEnd.3 murine brain endothelioma cells overexpressing transferrin receptors, by about 1.4-fold and 2.3-fold compared to that observed with the non-targeted dendriplex and naked DNA. This DNA uptake appeared to be optimal following 2h incubation with the treatment. In vivo, the intravenous injection of transferrin-bearing dendriplex more than doubled the gene expression in the brain compared to the unmodified dendriplex, while decreasing the non-specific gene expression in the lung. Gene expression was at least 3-fold higher in the brain than in any tested peripheral organs and was at its highest 24h following the injection of the treatments. These results suggest that transferrin-bearing polypropylenimine dendrimer is a highly promising gene delivery system to the brain. Copyright © 2014 Elsevier B.V. All rights reserved.

Dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic DNA damage in Huntington disease.

PubMed

Wang, Jiu-Qiang; Chen, Qian; Wang, Xianhua; Wang, Qiao-Chu; Wang, Yun; Cheng, He-Ping; Guo, Caixia; Sun, Qinmiao; Chen, Quan; Tang, Tie-Shan

2013-02-01

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

PubMed

Ahn, Seong Kyu; Cho, Pyo Yun; Na, Byoung-Kuk; Hong, Sung-Jong; Nam, Ho-Woo; Sohn, Woon-Mok; Ardelli, Bernadette F; Park, Yun-Kyu; Kim, Tong-Soo; Cha, Seok Ho

2016-01-01

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

PubMed

Sun, Yueying; Sun, Yuanyuan; Tian, Weimin; Liu, Chenghui; Gao, Kejian; Li, Zhengping

2018-02-07

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

Evaluating acetate metabolism for imaging and targeting in multiple myeloma

PubMed Central

Fontana, Francesca; Ge, Xia; Su, Xinming; Hathi, Deep; Xiang, Jingyu; Cenci, Simone; Civitelli, Roberto; Shoghi, Kooresh I.; Akers, Walter J.; D’avignon, Andre

2016-01-01

Purpose We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo. Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate-PET (positron emission tomography) to detect and quantitatively image myeloma treatment response in vivo. Experimental design Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. Results In vitro, NMR showed significant uptake of acetate by MMC, and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared to unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. Conclusions Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-acetate-PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-acetate-PET also detected response to therapy in vivo. Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate-PET is a promising imaging modality for MM. PMID:27486177

Methods to maximise recovery of environmental DNA from water samples.

PubMed

Hinlo, Rheyda; Gleeson, Dianne; Lintermans, Mark; Furlan, Elise

2017-01-01

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

Discrepancy between culture and DNA probe analysis for the detection of periodontal bacteria.

PubMed

van Steenbergen, T J; Timmerman, M F; Mikx, F H; de Quincey, G; van der Weijden, G A; van der Velden, U; de Graaff, J

1996-10-01

The purpose of this study was to compare a commercially available DNA probe technique with conventional cultural techniques for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque samples. Samples from 20 patients with moderate to severe periodontitis were evaluated at baseline and during a 15 months period of periodontal treatment. Paperpoints from 4 periodontal pockets per patient were forwarded to Omnigene for DNA probe analysis, and simultaneously inserted paperpoints from the same pockets were analyzed by standard culture techniques. In addition, mixed bacterial samples were constructed harbouring known proportions of 25 strains of A. actinomycetemcomitans, P. gingivalis and P. intermedia each. A relatively low concordance was found between both methods. At baseline a higher detection frequency was found for A. actinomycetemcomitans and P. gingivalis for the DNA probe technique; for P. intermedia the detection frequency by culture was higher. For A. actinomycetemcomitans, 21% of the culture positive samples was positive with the DNA probe. Testing the constructed bacterial samples with the DNA probe method resulted in about 16% false positive results for the 3 species tested. Furthermore, 40% of P. gingivalis strains were not detected by the DNA probe. The present data suggest that at least part of the discrepancies found between the DNA probe technique used and cultural methods are caused by false positive and false negative DNA probe results. Therefore, the value of this DNA probe method for the detection of periodontal pathogens is questionable.

Next-Generation Sequencing-Based Detection of Circulating Tumour DNA After Allogeneic Stem Cell Transplantation for Lymphoma

PubMed Central

Herrera, Alex F.; Kim, Haesook T.; Kong, Katherine A.; Faham, Malek; Sun, Heather; Sohani, Aliyah R.; Alyea, Edwin P.; Carlton, Victoria E.; Chen, Yi-Bin; Cutler, Corey S.; Ho, Vincent T.; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J.; Soiffer, Robert J.; Antin, Joseph H.; Armand, Philippe

2016-01-01

Summary Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3.7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% versus 84% in ctDNA-negative patients, p=0.033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3.9, p=0.003) and increased risk of relapse/progression (Hazard ratio 10.8, p=0.0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. PMID:27711974

EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: analysis of LUX-Lung 3 and 6

PubMed Central

Wu, Yi-Long; Sequist, Lecia V; Hu, Cheng-Ping; Feng, Jifeng; Lu, Shun; Huang, Yunchao; Li, Wei; Hou, Mei; Schuler, Martin; Mok, Tony; Yamamoto, Nobuyuki; O'Byrne, Kenneth; Hirsh, Vera; Gibson, Neil; Massey, Dan; Kim, Miyoung; Yang, James Chih-Hsin

2017-01-01

Background: In the Phase III LUX-Lung 3/6 (LL3/LL6) trials in epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma patients, we evaluated feasibility of EGFR mutation detection using circulating cell-free DNA (cfDNA) and prognostic and predictive utility of cfDNA positivity (cfDNA+). Methods: Paired tumour and blood samples were prospectively collected from randomised patients. Mutations were detected using cfDNA from serum (LL3) or plasma (LL6) by a validated allele-specific quantitative real-time PCR kit. Results: EGFR mutation detection rates in cfDNA were 28.6% (serum) and 60.5% (plasma). Mutation detection in blood was associated with advanced disease characteristics, including higher performance score, number of metastatic sites and bone/liver metastases, and poorer prognosis. In patients with common EGFR mutations, afatinib improved progression-free survival vs chemotherapy in cfDNA+ (LL3: HR, 0.35; P=0.0009; LL6: HR, 0.25; P<0.0001) and cfDNA− (LL3: HR, 0.46; P<0.0001; LL6: HR, 0.12; P<0.0001) cohorts. A trend towards overall survival benefit with afatinib was observed in cfDNA+ patients. Conclusions: Plasma cfDNA is a promising alternative to biopsy for EGFR testing. Detectable mutation in blood was associated with more advanced disease and poorer prognosis. Afatinib improved outcomes in EGFR mutation-positive patients regardless of blood mutation status. PMID:28006816

Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Xun; Liu, Guodong; Wang, Shengfu

2008-11-01

We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

New applications of CRISPR/Cas9 system on mutant DNA detection.

PubMed

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

[Current situation and prospect of breast cancer liquid biopsy].

PubMed

Zhou, B; Xin, L; Xu, L; Ye, J M; Liu, Y H

2018-02-01

Liquid biopsy is a diagnostic approach by analyzing body fluid samples. Peripheral blood is the most common sample. Urine, saliva, pleural effusion and ascites are also used. Now liquid biopsy is mainly used in the area of neoplasm diagnosis and treatment. Compared with traditional tissue biopsy, liquid biopsy is minimally invasive, convenient to sample and easy to repeat. Liquid biopsy mainly includes circulating tumor cells and circulating tumor DNA (ctDNA) detection. Detection of ctDNA requires sensitive and accurate methods. The progression of next-generation sequencing (NGS) and digital PCR promote the process of studies in ctDNA. In 2016, Nature published the result of whole-genome sequencing study of breast cancer. The study found 1 628 mutations of 93 protein-coding genes which may be driver mutations of breast cancer. The result of this study provided a new platform for breast cancer ctDNA studies. In recent years, there were many studies using ctDNA detection to monitor therapeutic effect and guide treatment. NGS is a promising technique in accessing genetic information and guiding targeted therapy. It must be emphasized that ctDNA detection using NGS is still at research stage. It is important to standardize ctDNA detection technique and perform prospective clinical researches. The time is not ripe for using ctDNA detection to guide large-scale breast cancer clinical practice at present.

Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

PubMed

Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

2016-08-24

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

PubMed

Gao, Ran; Lu, Dan-Feng; Cheng, Jin; Jiang, Yi; Jiang, Lan; Xu, Jian-Dong; Qi, Zhi-Mei

2016-12-15

An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Single DNA molecule detection using nanopipettes and nanoparticles.

PubMed

Karhanek, Miloslav; Kemp, Jennifer T; Pourmand, Nader; Davis, Ronald W; Webb, Chris D

2005-02-01

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haab, B.B.; Mathies, R.A.

A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative TO6 as they passed through the nearly 2-{mu}m diameter focused laser beam. Amplified photo-electron pulses from the photomultiplier are grouped into bins of 360-450 {mu}s in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were usedmore » to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The auto-correlation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of nearly 100 DNA molecules/band or better. 45 refs., 10 figs.« less

21 CFR 886.1670 - Ophthalmic isotope uptake probe.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., by a probe which is placed in close proximity to the eye, the uptake of a radioisotope (phosphorus 32) by tumors to detect tumor masses on, around, or within the eye. (b) Classification. Class II. ...

21 CFR 886.1670 - Ophthalmic isotope uptake probe.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., by a probe which is placed in close proximity to the eye, the uptake of a radioisotope (phosphorus 32) by tumors to detect tumor masses on, around, or within the eye. (b) Classification. Class II. ...

Potential utility of environmental DNA for early detection of Eurasian watermilfoil (Myriophyllum spicatum)

USGS Publications Warehouse

Newton, Jeremy; Sepulveda, Adam; Sylvester, K; Thum, Ryan

2016-01-01

Considering the harmful and irreversible consequences of many biological invasions, early detection of an invasive species is an important step toward protecting ecosystems (Sepulveda et al. 2012). Early detection increases the probability that suppression or eradication efforts will be successful because invasive populations are small and localized (Vander Zanden et al. 2010). However, most invasive species are not detected early because current tools have low detection probabilities when target species are rare and the sampling effort required to achieve acceptable detection capabilities with current tools is seldom tractable (Jerde et al. 2011). As a result, many invasive species go undetected until they are abundant and suppression efforts become costly. Novel DNA-based surveillance tools have recently revolutionized early detection abilities using environmental DNA (eDNA) present in the water (Darling and Mahon 2011, Bohmann et al. 2014). In brief, eDNA monitoring enables the identification of organisms from DNA present and collected in water samples. Aquatic and semiaquatic organisms release DNA contained in sloughed, damaged, or partially decomposed tissue and waste products into the water and molecular techniques allow this eDNA in the water column to be identified from simple and easy-tocollect water samples (Darling and Mahon 2011). Despite limited understanding of the production, persistence, and spread of DNA in water (Barnes et al. 2014), eDNA monitoring has been applied not only to invasive species (Jerde et al. 2011), but also to species that are rare, endangered, or highly elusive (Spear et al. 2014). However, most eDNA research and monitoring has focused on detection of invertebrates and vertebrates and less attentionhas been given to developing eDNA techniques for detecting aquatic invasive plants. Eurasian watermilfoil (EWM; Myriophyllum spicatum L.) is an invasive species for which improved early detection would be particularly helpful. Advanced EWM invasions have negative impacts on native biodiversity, recreational boating, fishing, and other types of aquatic tourism (e.g., Eiswerth et al. 2000). On a broader scale, EWM can also be harmful to man-made aquatic infrastructure, such as hydroelectric dams. If an EWM invasion can be detected in an early stage where eradication is still a possibility, many of these negative consequences can be limited or prevented altogether (e.g., Madsen et al. 2002). The purpose of this research was to develop and validate a traditional polymerase chain reaction (PCR) assay for the detection of pure and hybridized EWM DNA using both laboratory and field experiments. We performed a pilot experiment in outdoor tanks to determine the basic functionality and sensitivity of the assay. Following this initial test, we collected field samples from Michigan and Montana lakes with and without known EWM populations. Taken together, our findings suggest that eDNA techniques have potential to be a useful strategy for the early detection of EWM.

In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death.

PubMed

Ghosh, Ilika; Mukherjee, Amitava; Mukherjee, Anita

2017-05-01

Nanoremediation of soil, ground and surface water using nanoscale zerovalent iron particles (nZVI) has facilitated their direct environmental exposure posing ecotoxicological concerns. Numerous studies elucidate their phytotoxicity in terms of growth and their fate within the plant system. However, their potential genotoxicity and cytotoxicity mechanisms are not known in plants. This study encompasses the physico-chemical characterisation of two forms of nZVI (nZVI-1 and nZVI-2) with different surface chemistries and their influence on uptake, root morphology, DNA damage, oxidative stress and cell death in Allium cepa roots after 24 h. To our knowledge, this is the first report on the cyto-genotoxicity of nZVI in plants. The adsorption of nZVI on root surfaces caused root tip, epidermal and root hair damage as assessed by Scanning Electron Microscopy. nZVI-1, due to its colloidal destabilisation (low zeta potential, conductivity and high polydispersity index), smaller size and high uptake imparted enhanced DNA damage, chromosome/nuclear aberrations (CAs/NAs) and micronuclei formation compared to nZVI-2. Although nZVI-2 exhibited high zeta potential and conductivity, its higher dissolution and substantial uptake induced genotoxicity. nZVI incited the generation of reactive oxygen species (ROS) (hydrogen peroxide, superoxide and hydroxyl radicals) leading to membrane lipid peroxidation, electrolyte leakage and mitochondrial depolarisation. The inactivation of catalase and insignificant glutathione levels marked the onset of oxidative stress. Increased superoxide dismutase and guaiacol peroxidase enzyme activities, and proline content indicated the activation of antioxidant defence machinery to alleviate ROS. Moreover, ROS-mediated apoptotic and necrotic cell death occurred in both nZVI-1 and nZVI-2-treated roots. Our results open up further possibilities in the environmental safety appraisal of bare and modified nZVI in correlation with their physico-chemical characters. © The Author 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

PubMed Central

2012-01-01

Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains. Conclusion This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen. PMID:22309695

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

PubMed

Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2017-06-15

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R 2 =0.9984) between the fluorescent intensity and the target DNA concentration in the samples. Copyright © 2016. Published by Elsevier B.V.

Plasmonic SERS nanochips and nanoprobes for medical diagnostics and bio-energy applications

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Wang, Hsin-Neng; Crawford, Bridget M.; Fales, Andrew M.; Vo-Dinh, Tuan

2017-02-01

The development of rapid, easy-to-use, cost-effective, high accuracy, and high sensitive DNA detection methods for molecular diagnostics has been receiving increasing interest. Over the last five years, our laboratory has developed several chip-based DNA detection techniques including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). In these techniques, plasmonic surface-enhanced Raman scattering (SERS) Nanowave chips were functionalized with DNA probes for single-step DNA detection. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the Nanowave chip's gold surface. This distance change resulted in change in SERS intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized Nanowave chips and SERS signals were measured after 1h - 2h incubation. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost effective. The usefulness of the techniques for medical diagnostics was illustrated by the detection of genetic biomarkers for respiratory viral infection and of dengue virus 4 DNA.

Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

NASA Astrophysics Data System (ADS)

Heller, Michael J.; Krishnan, Raj; Sonnenberg, Avery

2010-08-01

The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

PubMed

Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

2015-12-15

We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. Copyright © 2015 Elsevier B.V. All rights reserved.

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

NASA Astrophysics Data System (ADS)

Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

2015-02-01

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening. Electronic supplementary information (ESI) available: Including detailed synthetic procedures of the dye and conjugate synthesis, as well as cellular uptake and inhibitor studies. See DOI: 10.1039/c4nr04467c

[Application of Liquid Biopsy for Lung Cancer Treatment.

PubMed

Mori, Shunsuke; Yatabe, Yasushi

2016-05-01

Liquid biopsy is defined as a non-invasive blood test that detects features of tumor cells, which are shed into the blood stream from the primary tumor and/or metastatic sites. This method is developing based on research on circulating tumor cells (CTCs) and the circulating free/fragments of tumor DNA (cfDNA). CfDNA can be detected in the absence of detectable CTCs, and has been shown to increase with the disease condition. The detection of cfDNA can be used for tumor genotyping, monitoring of the tumor burden, and monitoring minimal residual diseases, and recent results showed that cfDNA is a highly specific biomarker with intermediate sensitivity. Liquid biopsy with cfDNA is promising, and is becoming an alternative to re- biopsy. However, there are some caveats: it has not been elucidated which patients and tumor types can be accessed with cfDNA. Further research is warranted.

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

NASA Astrophysics Data System (ADS)

Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

2014-01-01

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

NASA Astrophysics Data System (ADS)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

NASA Astrophysics Data System (ADS)

Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.

2017-05-01

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

DNA Tumor Viruses and Cell Metabolism

PubMed Central

Mushtaq, Muhammad; Darekar, Suhas

2016-01-01

Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells. PMID:27034740

Lightning-triggered electroporation and electrofusion as possible contributors to natural horizontal gene transfer.

PubMed

Kotnik, Tadej

2013-09-01

Phylogenetic studies show that horizontal gene transfer (HGT) is a significant contributor to genetic variability of prokaryotes, and was perhaps even more abundant during the early evolution. Hitherto, research of natural HGT has mainly focused on three mechanisms of DNA transfer: conjugation, natural competence, and viral transduction. This paper discusses the feasibility of a fourth such mechanism--cell electroporation and/or electrofusion triggered by atmospheric electrostatic discharges (lightnings). A description of electroporation as a phenomenon is followed by a review of experimental evidence that electroporation of prokaryotes in aqueous environments can result in release of non-denatured DNA, as well as uptake of DNA from the surroundings and transformation. Similarly, a description of electrofusion is followed by a review of experiments showing that prokaryotes devoid of cell wall can electrofuse into hybrids expressing the genes of their both precursors. Under sufficiently fine-tuned conditions, electroporation and electrofusion are efficient tools for artificial transformation and hybridization, respectively, but the quantitative analysis developed here shows that conditions for electroporation-based DNA release, DNA uptake and transformation, as well as for electrofusion are also present in many natural aqueous environments exposed to lightnings. Electroporation is thus a plausible contributor to natural HGT among prokaryotes, and could have been particularly important during the early evolution, when the other mechanisms might have been scarcer or nonexistent. In modern prokaryotes, natural absence of the cell wall is rare, but it is reasonable to assume that the wall has formed during a certain stage of evolution, and at least prior to this, electrofusion could also have contributed to natural HGT. The concluding section outlines several guidelines for assessment of the feasibility of lightning-triggered HGT. © 2013 Elsevier B.V. All rights reserved.

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

NASA Astrophysics Data System (ADS)

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-01

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Maternal plasma DNA testing: experience of women counseled at a prenatal diagnosis center.

PubMed

O'Brien, Barbara M; Kloza, Edward M; Halliday, Jacquelyn V; Lambert-Messerlian, Geralyn M; Palomaki, Glenn E

2014-10-01

To evaluate the early introduction of circulating cell-free (ccf) DNA testing in a prenatal diagnosis center serving a statewide population. A retrospective chart review of patients at high aneuploidy risk counseled during the two 10-week periods that documents indication, risk, maternal age, insurance coverage, decisions, and reasoning behind that decision. Among the 299 included women, indication was advanced maternal age (17% with and 56% without an additional indication), positive serum screen (15%), and abnormal ultrasound (12%). Uptake increased from 10% to 17%, as did patient awareness of the test (4% to 14%). Women with lower copayments were more likely to complete testing (23% vs. 5%, p<0.001). Most women completing testing (75%) wanted to avoid an invasive procedure, while those declining cited testing would not change anything (47%), preferred diagnostic testing (16%), negative follow-up testing (20%), and cost/insurance issues (9%). One of 42 tests was positive (trisomy 21). Individual patient follow-up allows us to document ccfDNA-related patient decision-making. Nearly half of the women did not want further testing and one in seven preferred immediate diagnostic testing. Patient costs were a barrier to testing that, if avoided, could increase test uptake by 50% or more.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors.

PubMed

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-17

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

A paper-based device for double-stranded DNA detection with Zif268

NASA Astrophysics Data System (ADS)

Zhang, Daohong

2017-05-01

Here, a small analytical device was fabricated on both nitrocellulose membrane and filter paper, for the detection of biotinylated double-stranded DNA (dsDNA) from 1 nM. Zif268 was utilized for capturing the target DNA, which was a zinc finger protein that recognized only a dsDNA with specific sequence. Therefore, this detection platform could be utilized for PCR result detection, with the well-designed primers (interpolate both biotin and Zif268 binding sequence). The result of the assay could be recorded by a camera-phone, and analyzed with software. The whole assay finished within 1 hour. Due to the easy fabrication, operation and disposal of this device, this method can be employed in point-of-care detection or on-site monitoring.

Nonadiabatic tapered optical fiber sensor for measuring interaction nicotine with DNA

NASA Astrophysics Data System (ADS)

Zibaii, M. I.; Latifi, H.; Pourbeyram, H.; Gholami, M.; Taghipour, Z.; Saeedian, Z.; Hosseini, S. M.

2011-05-01

A nonadiabatic tapered optical fiber sensor was utilized for studying of bimolecular interactions including DNA-DNA and DNA-Drug interaction. This work presents a simple evanescent wave sensing system based on an interferometric approach, suitable to meet the requirements of lable-free sensor systems for detecting biomolecular interactions. We have demonstrated the measuring refractive index and the real time detection of interactions between biomolecules. Furthermore basic experiments were carried out, for detecting the hybridization of 25-mer DNA with an immobilized counterpart on the surface. The overall shift after the successful DNA hybridization was 9.5 nm. In this work, a new approach for studying DNA-drug interactions was successfully tested. Nicotine as a carcinogenic compound in cigarette smoke plays an important role in interaction with DNA. Different concentrations of nicotine were applied to observe the Longmuir interaction with DNA.

Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook

2014-09-03

A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulsemore » voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.« less

Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions

NASA Astrophysics Data System (ADS)

Kemp, David J.; Smith, Donald B.; Foote, Simon J.; Samaras, N.; Peterson, M. Gregory

1989-04-01

The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.

Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

PubMed

Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

2012-09-15

A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery.

PubMed

Kimura, Tsuyoshi; Nibe, Yoichi; Funamoto, Seiichi; Okada, Masahiro; Furuzono, Tsutomu; Ono, Tsutomu; Yoshizawa, Hidekazu; Fujisato, Toshiya; Nam, Kwangwoo; Kishida, Akio

2011-01-01

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.

Comparison of oral fluid collection methods for the molecular detection of hepatitis B virus.

PubMed

Portilho, M M; Mendonça, Acf; Marques, V A; Nabuco, L C; Villela-Nogueira, C A; Ivantes, Cap; Lewis-Ximenez, L L; Lampe, E; Villar, L M

2017-11-01

This study aims to compare the efficiency of four oral fluid collection methods (Salivette, FTA Card, spitting and DNA-Sal) to detect HBV DNA by qualitative PCR. Seventy-four individuals (32 HBV reactive and 42 with no HBV markers) donated serum and oral fluid. In-house qualitative PCR to detect HBV was used for both samples and commercial quantitative PCR for serum. HBV DNA was detected in all serum samples from HBV-infected individuals, and it was not detected in control group. HBV DNA from HBV group was detected in 17 samples collected with Salivette device, 16 samples collected by FTA Card device, 16 samples collected from spitting and 13 samples collected by DNA-Sal device. Samples that corresponded to a higher viral load in their paired serum sample could be detected using all oral fluid collection methods, but Salivette collection device yielded the largest numbers of positive samples and had a wide range of viral load that was detected. It was possible to detect HBV DNA using all devices tested, but higher number of positive samples was observed when samples were collected using Salivette device, which shows high concordance to viral load observed in the paired serum samples. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

A feasibility study of colorectal cancer diagnosis via circulating tumor DNA derived CNV detection.

PubMed

Molparia, Bhuvan; Oliveira, Glenn; Wagner, Jennifer L; Spencer, Emily G; Torkamani, Ali

2018-01-01

Circulating tumor DNA (ctDNA) has shown great promise as a biomarker for early detection of cancer. However, due to the low abundance of ctDNA, especially at early stages, it is hard to detect at high accuracies while keeping sequencing costs low. Here we present a pilot stage study to detect large scale somatic copy numbers variations (CNVs), which contribute more molecules to ctDNA signal compared to point mutations, via cell free DNA sequencing. We show that it is possible to detect somatic CNVs in early stage colorectal cancer (CRC) patients and subsequently discriminate them from normal patients. With 25 normal and 24 CRC samples, we achieve 100% specificity (lower bound confidence interval: 86%) and ~79% sensitivity (95% confidence interval: 63% - 95%,), though the performance should be considered with caution given the limited sample size. We report a lack of concordance between the CNVs detected via cfDNA sequencing and CNVs identified in parent tissue samples. However, recent findings suggest that a lack of concordance is expected for CNVs in CRC because of their sub-clonal nature. Finally, the CNVs we detect very likely contribute to cancer progression as they lie in functionally important regions, and have been shown to be associated with CRC specifically. This study paves the path for a larger scale exploration of the potential of CNV detection for both diagnoses and prognoses of cancer.

Epidemiologic and Molecular Relationship Between Vaccine Manufacture and Autism Spectrum Disorder Prevalence.

PubMed

Deisher, Theresa A; Doan, Ngoc V; Koyama, Kumiko; Bwabye, Sarah

2015-01-01

To assess the public health consequences of fetal cell line manufactured vaccines that contain residual human fetal DNA fragments utilizing laboratory and ecological approaches including statistics, molecular biology and genomics. MMR coverage and autism disorder or autism spectrum disorder prevalence data for Norway, Sweden and the UK were obtained from public and government websites as well as peer reviewed published articles. Biologically, the size and quantity of the contaminating fetal DNA in Meruvax II and Havrix as well as the propensity of various cell lines for cellular and nuclear uptake of primitive human DNA fragments were measured and quantified using gel electrophoresis, fluorescence microscopy and fluorometry. Lastly, genomic analysis identified the specific sites where fetal DNA fragment integration into a child's genome is most likely to occur. The average MMR coverage for the three countries fell below 90% after Dr. Wakefield's infamous 1998 publication but started to recover slowly after 2001 until reaching over 90% coverage again by 2004. During the same time period, the average autism spectrum disorder prevalence in the United Kingdom, Norway and Sweden dropped substantially after birth year 1998 and gradually increased again after birth year 2000. Average single stranded DNA and double stranded DNA in Meruvax II were 142.05 ng/vial and 35.00 ng/vial, respectively, and 276.00 ng/vial and 35.74 ng/vial in Havrix respectively. The size of the fetal DNA fragments in Meruvax II was approximately 215 base pairs. There was spontaneous cellular and nuclear DNA uptake in HFF1 and NCCIT cells. Genes that have been linked to autism (autism associated genes; AAGs) have a more concentrated susceptibility for insults to genomic stability in comparison to the group of all genes contained within the human genome. Of the X chromosome AAGs, 15 of 19 have double strand break motifs less than 100 kilobases away from the center of a meiotic recombination hotspot located within an exon. Vaccines manufactured in human fetal cell lines contain unacceptably high levels of fetal DNA fragment contaminants. The human genome naturally contains regions that are susceptible to double strand break formation and DNA insertional mutagenesis. The "Wakefield Scare" created a natural experiment that may demonstrate a causal relationship between fetal cell-line manufactured vaccines and ASD prevalence.

Colonization of citrus seed coats by 'Candidatus Liberibacter asiaticus': implications for seed transmission of the bacterium.

PubMed

Hilf, Mark E

2011-10-01

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.

Uptake of a fluorescent L-glucose derivative 2-NBDLG into three-dimensionally accumulating insulinoma cells in a phloretin-sensitive manner.

PubMed

Sasaki, Ayako; Nagatomo, Katsuhiro; Ono, Koki; Yamamoto, Toshihiro; Otsuka, Yuji; Teshima, Tadashi; Yamada, Katsuya

2016-01-01

Of two stereoisomers of glucose, only D- and not L-glucose is abundantly found in nature, being utilized as an essential fuel by most organisms. The uptake of D-glucose into mammalian cells occurs through glucose transporters such as GLUTs, and this process has been effectively monitored by a fluorescent D-glucose derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) at the single cell level. However, since fluorescence is an arbitrary measure, we have developed a fluorescent analog of L-glucose 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), as a negative control substrate for more accurately identifying the stereoselectivity of the uptake. Interestingly, a small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (≳ 10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage (≲ 6 DIV). The 2-NBDLG uptake was persistently observed in the presence of a GLUT inhibitor cytochalasin B. Neither D- nor L-glucose in 50 mM abolished the uptake. No significant inhibition was detected by inactivating sodium/glucose cotransporters (SGLTs) with Na(+)-free condition. To our surprise, the 2-NBDLG uptake was totally inhibited by phloretin, a broad spectrum inhibitor against transporters/channels including GLUTs and aquaporins. From these, a question might be raised if non-GLUT/non-SGLT pathways participate in the 2-NBDLG uptake into spheroid-forming MIN6 insulinoma. It might also be worthwhile investigating whether 2-NBDLG can be used as a functional probe for detecting cancer, since the nuclear heterogeneity is among critical features of malignancy.

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed Frogs and Idaho Giant Salamanders

PubMed Central

Goldberg, Caren S.; Pilliod, David S.; Arkle, Robert S.; Waits, Lisette P.

2011-01-01

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research. PMID:21818382

Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders.

PubMed

Goldberg, Caren S; Pilliod, David S; Arkle, Robert S; Waits, Lisette P

2011-01-01

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

Comparison between Urine and Cervical Samples for HPV DNA Detection and Typing in Young Women in Colombia.

PubMed

Cómbita, Alba Lucía; Gheit, Tarik; González, Paula; Puerto, Devi; Murillo, Raúl Hernando; Montoya, Luisa; Vorsters, Alex; Van Keer, Severien; Van Damme, Pierre; Tommasino, Massimo; Hernández-Suárez, Gustavo; Sánchez, Laura; Herrero, Rolando; Wiesner, Carolina

2016-09-01

Urine sampling for HPV DNA detection has been proposed as an effective method for monitoring the impact of HPV vaccination programs; however, conflicting results have been reported. The goal of this study was to evaluate the performance of optimized urine HPV DNA testing in women aged 19 to 25 years. Optimization process included the use of first void urine, immediate mixing of urine with DNA preservative, and the concentration of all HPV DNA, including cell-free DNA fragments. Urine and cervical samples were collected from 535 young women attending cervical screening at health centers from two Colombian cities. HPV DNA detection and genotyping was performed using an HPV type-specific multiplex genotyping assay, which combines multiplex polymerase chain reaction with bead-based Luminex technology. Concordance between HPV DNA detection in urine and cervical samples was determined using kappa statistics and McNemar tests. The accuracy of HPV DNA testing in urine samples was evaluated measuring sensitivity and specificity using as reference the results obtained from cervical samples. Statistical analysis was performed using STATA11.2 software. The findings revealed an overall HPV prevalence of 60.00% in cervical samples and 64.72% in urine samples, HPV-16 being the most frequent HPV type detected in both specimens. Moreover, our results indicate that detection of HPV DNA in first void urine provides similar results to those obtained with cervical samples and can be used to monitor HPV vaccination trials and programs as evidenced by the substantial concordance found for the detection of the four vaccine types. Cancer Prev Res; 9(9); 766-71. ©2016 AACR. ©2016 American Association for Cancer Research.

Fine-tuning for the tropics: application of eDNA technology for invasive fish detection in tropical freshwater ecosystems.

PubMed

Robson, Heather L A; Noble, Tansyn H; Saunders, Richard J; Robson, Simon K A; Burrows, Damien W; Jerry, Dean R

2016-07-01

Invasive species pose a major threat to aquatic ecosystems. Their impact can be particularly severe in tropical regions, like those in northern Australia, where >20 invasive fish species are recorded. In temperate regions, environmental DNA (eDNA) technology is gaining momentum as a tool to detect aquatic pests, but the technology's effectiveness has not been fully explored in tropical systems with their unique climatic challenges (i.e. high turbidity, temperatures and ultraviolet light). In this study, we modified conventional eDNA protocols for use in tropical environments using the invasive fish, Mozambique tilapia (Oreochromis mossambicus) as a detection model. We evaluated the effects of high water temperatures and fish density on the detection of tilapia eDNA, using filters with larger pores to facilitate filtration. Large-pore filters (20 μm) were effective in filtering turbid waters and retaining sufficient eDNA, whilst achieving filtration times of 2-3 min per 2-L sample. High water temperatures, often experienced in the tropics (23, 29, 35 °C), did not affect eDNA degradation rates, although high temperatures (35 °C) did significantly increase fish eDNA shedding rates. We established a minimum detection limit for tilapia (1 fish/0.4 megalitres/after 4 days) and found that low water flow (3.17 L/s) into ponds with high fish density (>16 fish/0.4 megalitres) did not affect eDNA detection. These results demonstrate that eDNA technology can be effectively used in tropical ecosystems to detect invasive fish species. © 2016 John Wiley & Sons Ltd.

Molecular detection of vertebrates in stream water: A demonstration using rocky mountain tailed frogs and Idaho giant salamanders

USGS Publications Warehouse

Goldberg, C.S.; Pilliod, D.S.; Arkle, R.S.; Waits, L.P.

2011-01-01

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

Protective Properties of Radio-Chemoresistant Glioblastoma Stem Cell Clones Are Associated with Metabolic Adaptation to Reduced Glucose Dependence

PubMed Central

Yamada, Kazunari; Tso, Jonathan L.; Menjivar, Jimmy C.; Tian, Jane Y.; Yong, William H.; Schaue, Dörthe; Mischel, Paul S.; Cloughesy, Timothy F.; Nelson, Stanley F.; Liau, Linda M.; McBride, William; Tso, Cho-Lea

2013-01-01

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma. PMID:24260384

Enhanced photoelectrochemical DNA sensor based on TiO2/Au hybrid structure.

PubMed

Liu, Xing-Pei; Chen, Jing-Shuai; Mao, Chang-Jie; Niu, He-Lin; Song, Ji-Ming; Jin, Bao-Kang

2018-05-23

A novel enhanced photoelectrochemical DNA sensor, based on a TiO 2 /Au hybrid electrode structure, was developed to detect target DNA. The sensor was developed by successively modifying fluorine-tin oxide (FTO) electrodes with TiO 2 nanoparticles, gold (Au) nanoparticles, hairpin DNA (DNA1), and CdSe-COOH quantum dots (QDs), which acted as signal amplification factors. In the absence of target DNA, the incubated DNA1 hairpin and the CdSe-COOH QDs were in close contact with the TiO 2 /Au electrode surface, leading to an enhanced photocurrent intensity due to the sensitization effect. After incubation of the modified electrode with the target DNA, the hairpin DNA changed into a double helix structure, and the CdSe QDs moved away from the TiO 2 /Au electrode surface, leading to a decreased sensitization effect and photoelectrochemical signal intensity. This novel DNA sensor exhibited stable, sensitive and reproducible detection of DNA from 0.1 μM to 10 fM, with a lower detection limit of 3 fM. It provided good specificity, reproducibility, stability and is a promising strategy for the detection of a variety of other DNA targets, for early clinical diagnosis of various diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis.

PubMed

Peeters, Dominique; Peters, Iain R; Helps, Chris R; Dehard, Sandrine; Day, Michael J; Clercx, Cécile

2008-04-01

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.

Analytical Devices Based on Direct Synthesis of DNA on Paper.

PubMed

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Fungal DNA in hotel rooms in Europe and Asia--associations with latitude, precipitation, building data, room characteristics and hotel ranking.

PubMed

Norbäck, Dan; Cai, Gui-Hong

2011-10-01

There is little information on the indoor environment in hotels. Analysis of fungal DNA by quantitative PCR (qPCR) is a new method which can detect general and specific sequences. Dust was collected through swab sampling of door frames in 69 hotel rooms in 20 countries in Europe and Asia (2007-2009). Five sequences were detected by qPCR: total fungal DNA, Aspergillus and Penicillium DNA (Asp/Pen DNA), Aspergillus versicolor (A. versicolor DNA), Stachybotrys chartarum (S. chartarum DNA) and Streptomyces spp. (Streptomyces DNA). Associations were analysed by multiple linear regression. Total fungal DNA (GM = 1.08 × 10(8) cell equivalents m(-2); GSD = 6.36) and Asp/Pen DNA (GM = 1.79 × 10(7) cell equivalents m(-2); GSD = 10.12) were detected in all rooms. A. versicolor DNA, S. chartarum DNA and Streptomyces DNA were detected in 84%, 28% and 47% of the samples. In total, 20% of the rooms had observed dampness/mould, and 30% had odour. Low latitude (range 1.5-64.2 degrees) was a predictor of Asp/Pen DNA. Seaside location, lack of mechanical ventilation, and dampness or mould were other predictors of total fungal DNA and Asp/Pen DNA. Hotel ranking (Trip Advisor) or self-rated quality of the interior of the hotel room was a predictor of total fungal DNA, A. versicolor DNA and Streptomyces DNA. Odour was a predictor of S. chartarum DNA. In conclusion, fungal DNA in swab samples from hotel rooms was related to latitude, seaside location, ventilation, visible dampness and indoor mould growth. Hotels in tropical areas may have 10-100 times higher levels of common moulds such as Aspergillus and Penicillium species, as compared to a temperate climate zone.

Moving environmental DNA methods from concept to practice for monitoring aquatic macroorganisms

USGS Publications Warehouse

Goldberg, Caren S.; Strickler, Katherine M.; Pilliod, David S.

2015-01-01

The discovery that macroorganisms can be detected from their environmental DNA (eDNA) in aquatic systems has immense potential for the conservation of biological diversity. This special issue contains 11 papers that review and advance the field of eDNA detection of vertebrates and other macroorganisms, including studies of eDNA production, transport, and degradation; sample collection and processing to maximize detection rates; and applications of eDNA for conservation using citizen scientists. This body of work is an important contribution to the ongoing efforts to take eDNA detection of macroorganisms from technical breakthrough to established, reliable method that can be used in survey, monitoring, and research applications worldwide. While the rapid advances in this field are remarkable, important challenges remain, including consensus on best practices for collection and analysis, understanding of eDNA diffusion and transport, and avoidance of inhibition in sample collection and processing. Nonetheless, as demonstrated in this special issue, eDNA techniques for research and monitoring are beginning to realize their potential for contributing to the conservation of biodiversity globally.

Integrated digital error suppression for improved detection of circulating tumor DNA

PubMed Central

Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.

2016-01-01

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

PubMed Central

Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

2011-01-01

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

Detection of incidental colorectal pathology on positron emission tomography/computed tomography.

PubMed

Mui, Milton; Akhurst, Timothy; Warrier, Satish K; Lynch, A Craig; Heriot, Alexander G

2018-03-01

Positron emission tomography/computed tomography (PET/CT) is an important modality in cancer imaging. With its increasing availability and use, it is not uncommon to detect incidental focal colorectal 18 F-FDG uptake which poses a diagnostic challenge, as they may be associated with malignant or pre-malignant colorectal lesions. The aim of our study is to determine the proportion of these findings which represents true pathology. Patients with incidental focal colorectal 18 F-FDG uptake on PET/CT who subsequently underwent colonoscopy between January 2002 to September 2013 were identified from a prospective database in a tertiary referral centre. PET/CT results were correlated with colonoscopy and pathology results in these patients. Positive predictive values (PPVs) and 95% confidence intervals (CIs) of PET/CT in the detection of incidental colorectal pathology were calculated. A total of 148 patients (92 men and 56 women), with a mean age 73 years (range of 36 to 93 years) were included in the study. A total of 170 foci of colorectal 18 F-FDG uptake were detected on PET/CT. Of these, 101 foci corresponded to a malignant or pre-malignant lesion (PPV 59%; 95% CI: 52-67%). On a per-patient analysis, 93 patients had at least one focus of colorectal 18 F-FDG uptake which corresponded to a pre-malignant or malignant lesion (PPV 63%; 95% CI: 54-71%). Focal colorectal 18 F-FDG uptake on PET/CT is associated with a significant proportion of malignant or pre-malignant lesions. Further evaluation with colonoscopy is recommended. © 2016 Royal Australasian College of Surgeons.

Nanoplasmonic biosensor: detection and amplification of dual bio-signatures of circulating tumor DNA.

PubMed

Nguyen, Anh H; Sim, Sang Jun

2015-05-15

Circulating tumor DNA (ctDNA) bearing tumor-specific mutation and methylation are promising biomarkers for noninvasive cancer assessment. However, existing methods for ctDNA detection are restricted to genetic mutations. Recently, nanoplasmonics has emerged as a platform for one-step dual detection with high sensitivity and specificity. Here we present a strategy for ultrasensitive detection of tumor-specific mutations (E542K and E545K) and methylation of ctDNA of PIK3CA gene based on localized surface plasmon resonance (LSPR) and the coupling plasmon mode of gold nanoparticles (AuNPs). Peptide nucleic acids (PNA) is used as a probe to capture and enrich the 69-bp PIK3CA ctDNA. The exposure of PNA-probed AuNPs to 200 fM ctDNA generates LSPR-peak shift of 4.3 nm, corresponding to the primary response. Immunogold colloids are exploited as methylation detectors and plasmon coupling based enhancement for secondary response. LSPR-peak shifted from 4.3 nm to 11.4 nm upon the immunogold colloids binding to two methylcytosines (mCpG), which is an approximately 107% increase, compared to that of the primary response. This enhancement leads to four times (~50 fM) improvement of sensitivity and because of two mCpG sites, ctDNA was detected. These results demonstrate that the sensor can simultaneously detect the hot-spot mutation and epigenetic changes on the ctDNA. Promisingly, other specific-tumor mutants and epigenetic changes can be detected at low concentration with this platform. Copyright © 2014 Elsevier B.V. All rights reserved.

Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

PubMed

Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

2016-07-01

As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Fabrication of an electrochemical DNA-based biosensor for Bacillus cereus detection in milk and infant formula.

PubMed

Izadi, Zahra; Sheikh-Zeinoddin, Mahmoud; Ensafi, Ali A; Soleimanian-Zad, Sabihe

2016-06-15

This paper describes fabrication of a DNA-based Au-nanoparticle modified pencil graphite electrode (PGE) biosensor for detection of Bacillus cereus, causative agent of two types of food-borne disease, i.e., emetic and diarrheal syndrome. The sensing element of the biosensor was comprised of gold nanoparticles (GNPs) self-assembled with single-stranded DNA (ssDNA) of nheA gene immobilized with thiol linker on the GNPs modified PGE. The size, shape and dispersion of the GNPs were characterized by field emission scanning electron microscope (FESEM). Detection of B. cereus was carried out based on an increase in the charge transfer resistance (Rct) of the biosensor due to hybridization of the ss-DNA with target DNA. An Atomic force microscope (AFM) was used to confirm the hybridization. The biosensor sensitivity in pure cultures of B. cereus was found to be 10(0) colony forming units per milliliter (CFU/mL) with a detection limit of 9.4 × 10(-12) mol L(-1). The biosensor could distinguish complementary from mismatch DNA sequence. The proposed biosensor exhibited a rapid detection, low cost, high sensitivity to bacterial contamination and could exclusively and specifically detect the target DNA sequence of B. cereus from other bacteria that can be found in dairy products. Moreover, the DNA biosensor exhibited high reproducibility and stability, thus it may be used as a suitable biosensor to detect B. cereus and to become a portable system for food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Value of FDG PET in the assessment of patients with multiple myeloma.

PubMed

Bredella, Miriam A; Steinbach, Lynne; Caputo, Gary; Segall, George; Hawkins, Randall

2005-04-01

Our objective was to evaluate if whole-body PET with FDG is able to detect bone marrow involvement in patients with multiple myeloma and to assess its appearance and distribution pattern. Seventeen whole-body FDG PET scans were performed in 13 patients with multiple myeloma. Four patients were referred for evaluation of extent of disease pretherapy and nine patients were referred for assessment of therapy response (chemotherapy, radiation therapy, bone marrow transplant). FDG PET images were evaluated for distribution and uptake pattern. Standardized uptake values were obtained to quantify FDG uptake. Results of other imaging examinations (MRI, CT, radiography), laboratory data, biopsies, and the clinical course were used for verification of detected lesions. FDG PET was able to detect medullary involvement of multiple myeloma. There were two false-negative results. In one patient, the radiographic skeletal survey showed subcentimeter lytic lesions within the ribs that were not detected on FDG PET and in the other patient, a lytic lesion detected on radiographs showed only mildly increased FDG uptake that was not identified prospectively. There was one false-positive FDG PET result in a patient who had undergone radiation therapy 3 weeks before PET. FDG PET was helpful in differentiating between posttherapeutic changes and residual/recurrent tumor and in assessing response to therapy. FDG PET resulted in upstaging of disease in four patients, which influenced subsequent management and prognosis. Sensitivity of FDG PET in detecting myelomatous involvement was 85% and specificity was 92%. FDG PET is able to detect bone marrow involvement in patients with multiple myeloma. FDG PET is useful in assessing extent of disease at time of initial diagnosis, contributing to staging that is more accurate. FDG PET is also useful for evaluating therapy response.

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

PubMed

Bai, Jinhe; Baldwin, Elizabeth; Liao, Hui-Ling; Zhao, Wei; Kostenyuk, Igor; Burns, Jacqueline; Irey, Mike

2013-10-02

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.

Highly sensitive DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization

PubMed Central

Yu, Xu; Zhang, Zhi-Ling; Zheng, Si-Yang

2014-01-01

A novel highly sensitive colorimetric assay for DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization was established. The DNA modified superparamagnetic beads were demonstrated to capture and enrich the target DNA in the hybridization buffer or human plasma. The hybridization chain reaction and enzyme-induced silver metallization on the gold nanoparticles were used as cascade signal amplification for the detection of target DNA. The metalization of silver on the gold nanoparticles induced a significant colour change from red to yellow until black depending on the concentration of the target DNA, which could be recognized by naked eyes. This method showed a good specificity for the target DNA detection, with the capabilty to discriminate single-base-pair mismatched DNA mutation (single nucleotide polymorphism). Meanwhile, this approach exhibited an excellent anti-interference capability with the convenience of the magentic seperation and washing, which enabled its usage in complex biological systems such as human blood plasma. As an added benefit, the utilization of hybridization chain reaction and enzyme-induced metallization improved detection sensitivity down to 10 pM, which is about 100-fold lower than that of traditional unamplified homogeneous assays. PMID:25500528

SIMSISH Technique Does Not Alter the Apparent Isotopic Composition of Bacterial Cells

PubMed Central

Chapleur, Olivier; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Mazéas, Laurent; Bouchez, Théodore

2013-01-01

In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific 13C uptake during labelled methanol anaerobic degradation. PMID:24204855

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tockman, M.S.

The author evaluated a synthetic porphyrin, 5, 10, 15, 20 tetrakis (4-carboxyphenyl) porphene (H{sub 2}TCPP) as a marker of carcinogenesis. H{sub 2}TCPP was compared with two other carcinogenesis markers evaluated in the laboratory for their ability to detect exfoliated sputum cells undergoing transformation to lung cancer. In the present project the authors first established optimal conditions for cultured neoplastic and non-neoplastic (sputum) cells to take up H{sub 2}TCPP. This was accomplished using spectrofluorimetry and video-enhanced fluorescent microscopy to maximize H{sub 2}TCPP auto-fluorescence across a matrix of substrate conditions, including; reagent concentration, incubation time, temperature, and pH. The second aim wasmore » to validate H{sub 2}TCPP on clinical material obtained from subjects monitored in advance of clinical cancer and link those marker results with subsequent histologic confirmation of disease. This was accomplished by applying H{sub 2}TCPP to sputum specimens archived by the Frost Center at Johns Hopkins which maintains a record of the clinical course and long-term follow-up for the patients from whom the specimens were obtained. The authors have used fluorescent immunostaining and flow cytometry to compare uptake of these cytoplasmic Mabs to that of a potential new marker of carcinogenesis, 5, 10, 15, 20 tetrakis (4 carboxyphenyl) porphene (H{sub 2}TCPP). The nuclear uptake of H{sub 2}TCPP was compared to a standard quantitative fluorescent DNA marker (7-AAD).« less

Nanoscale Formulations and Diagnostics With Their Recent Trends: A Major Focus of Future Nanotechnology.

PubMed

Mukherjee, Biswajit; Dutta, Lopamudra; Mondal, Laboni; Dey, Niladri Shekhar; Chakraborty, Samrat; Maji, Ruma; Shaw, Tapan Kumar

2015-01-01

Nanomedicine is an emerging and rapidly growing field, possibly exploring for high expectation to healthcare. Nanoformulations have been designed to overcome challenges due to the development and fabrication of nanostructures. Unique size-dependent properties of nanoformulations make them superior and indispensable in many areas of human activity. Nano drug delivery systems are formulated and engineered to carry and deliver a number of substances in a targeted and controlled way. The vision of nanocarriers can be designed that will serve a dual purpose, allowing both treatment and diagnosis to be contained in an 'all-in-one' package. Nanoscale drugdelivery systems efficiently regulate the release, pharmacokinetics, pharmacodynamics, solubility, immunocompatibility, cellular uptake and biodistribution of chemical entities (drug). Their cellular uptake takes place by various mechanisms such as micropinocytosis, phagocytosis and receptor mediated endocytosis. These phenomena cause longer retention in blood circulation resulting in the release of the encapsulated materials in a sustained manner thus minimize the plasma fluctuations and toxic side effects. In this manner, the therapeutic index of conventional pharmaceuticals is efficiently increased. They can be used to deliver both micro and macro biomolecules such as peptides, proteins, plasmid DNA and synthetic oligodeoxynucleotides. In this present review, several recent developing and modifying nano-products for the detection, analysis, and treatment of diseases with their US and world patents along with various diagnostic kits have been discussed.

Evolved osmotolerant Escherichia coli mutants frequently exhibit defective N-acetylglucosamine catabolism and point mutations in cell shape-regulating protein MreB.

PubMed

Winkler, James D; Garcia, Carlos; Olson, Michelle; Callaway, Emily; Kao, Katy C

2014-06-01

Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

[Detection of KRAS mutation in colorectal cancer patients' cfDNA with droplet digital PCR].

PubMed

Luo, Yuwen; Li, Yao

2018-03-25

This study aims to develop a new method for the detection of KRAS mutations related to colorectal cancer in cfDNA, and to evaluate the sensitivity and accuracy of the detection. We designed a method of cfDNA based KRAS detection by droplets digital PCR (ddPCR). The theoretical performance of the method is evaluated by reference standard and compared to the ARMS PCR method. Two methods, ddPCR and qPCR, were successfully established to detect KRAS wild type and 7 mutants. Both methods were validated using plasmid standards and actual samples. The results were evaluated by false positive rate, linearity, and limit of detection. Finally, 52 plasma cfDNA samples from patients and 20 samples from healthy people were tested, the clinical sensitivity is 97.64%, clinical specificity is 81.43%. ddPCR method shows higher performance than qPCR. The LOD of ddPCR method reached single digits of cfDNA copies, it can detect as low as 0.01% to 0.04% mutation abundance.

Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction.

PubMed

Yu, Luxin; Wu, Wei; Chen, Junhua; Xiao, Zhuo; Ge, Chenchen; Lie, Puchang; Fang, Zhiyuan; Chen, Lingbo; Zhang, Ya; Zeng, Lingwen

2013-12-07

We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.

A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection

NASA Astrophysics Data System (ADS)

Patel, Manoj Kumar; Ali, Md. Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, Abdulaziz A.; Fouad, H.; Ansari, Z. A.; Ansari, S. G.; Malhotra, Bansi D.

2015-11-01

Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R2) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

PubMed Central

Coldwell, Kate E.; Cutts, Suzanne M.; Ognibene, Ted J.; Henderson, Paul T.; Phillips, Don R.

2008-01-01

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/104 bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [14C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [14C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/107 bp (∼1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues. PMID:18632763

The value of the first trimester ultrasound in the era of cell free DNA screening.

PubMed

Rao, Rashmi R; Valderramos, Stephanie G; Silverman, Neil S; Han, Christina S; Platt, Lawrence D

2016-12-01

To describe the clinically relevant findings detected by the first trimester ultrasound (FTU) and to determine the additional value of the FTU compared to cell free DNA (cfDNA) alone. Retrospective cohort study of patients undergoing a FTU at a maternal-fetal medicine referral practice. Fetal, gynecologic, and placental findings detected by ultrasound were analyzed with available cfDNA and diagnostic testing results. A subgroup analysis of positive ultrasound findings and cfDNA results was performed to assess the additional benefit of ultrasound evaluation in FT prenatal screening. There were 1906 FTU between 1 October 2013 and 1 October 2014. CfDNA results were available for 959 (50%) patients. FTU detected: 42 fetal (2.2%), 286 gynecologic (15.0%), and 317 placental (16.6%) findings. CfDNA results were discordant with invasive testing results in 8/61 cases (13%) and with ultrasound findings in 18/42 (42%) cases. There were six false positive and two false negative cfDNA results confirmed by diagnostic testing. Subgroup analysis revealed that cfDNA as the sole method of prenatal screening in the FT would miss 95% of the fetal findings detected with ultrasound. The comprehensive FTU provides valuable clinical information about fetal and maternal anatomy that cannot be detected with cfDNA alone. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less