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A magnetic bead-based method for concentrating DNA from human urine for downstream detection.
Bordelon H, Russ PK, Wright DW, Haselton FR
(2013) PLoS One 8: e68369
MeSH Terms: Adsorption, Base Sequence, Buffers, DNA, Freeze Drying, Humans, Kinetics, Limit of Detection, Magnetics, Microspheres, Molecular Weight, Polymerase Chain Reaction, Reagent Kits, Diagnostic, Silicon Dioxide, Specimen Handling
Show Abstract · Added May 27, 2014
Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.
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15 MeSH Terms
Low-resource method for extracting the malarial biomarker histidine-rich protein II to enhance diagnostic test performance.
Davis KM, Swartz JD, Haselton FR, Wright DW
(2012) Anal Chem 84: 6136-42
MeSH Terms: Amino Acid Motifs, Antigens, Protozoan, Biomarkers, Buffers, Chemical Fractionation, Humans, Limit of Detection, Malaria, Protozoan Proteins
Show Abstract · Added May 27, 2014
We have demonstrated the utility of a self-contained extraction device for the selective isolation, purification, and concentration of the malaria diagnostic protein biomarker Plasmodium falciparum histidine-rich protein II (pfHRPII) from human plasma and whole blood. The extraction cassette consists of a small-diameter tube containing a series of preloaded processing solutions separated by mineral oil valves. Nickel(II) nitrilotriacetic acid-functionalized magnetic particles are added to a parasite-spiked sample contained within the loading chamber of the device for capture of pfHRPII. The biomarker-bound magnetic particles are then entrained by an external magnetic field and transported through three wash solutions. Processing removes sample interfering agents, and the biomarker target is concentrated in the final chamber for subsequent analysis. At parasitemias of 200 parasites/μL, purification and concentration of pfHRPII with extraction efficiencies in excess of 70% total protein target are achieved. The concentration of nonspecific protein interfering agents was reduced by more than 2 orders of magnitude in the final extracted sample without the need for hours of processing time and specialized laboratory equipment. We have demonstrated an application of this low-resource technology by coupling extraction and concentration of pfHRPII within the cassette to a commonly employed rapid diagnostic test. Sample preprocessing improved the visual limit of detection of this test by over 8-fold, suggesting that the combination of both low-resource technologies could prove to be useful in malaria eradication efforts.
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9 MeSH Terms
Solution protein digest.
Link AJ, LaBaer J
(2011) Cold Spring Harb Protoc 2011: pdb.prot5569
MeSH Terms: Buffers, Protein Denaturation, Proteins, Solutions, Tandem Mass Spectrometry, Trypsin
Added February 20, 2015
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6 MeSH Terms
Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin.
Griesser M, Pistis V, Suzuki T, Tejera N, Pratt DA, Schneider C
(2011) J Biol Chem 286: 1114-24
MeSH Terms: Animals, Antineoplastic Agents, Buffers, Cell Transformation, Neoplastic, Cells, Cultured, Curcumin, Cyclooxygenase 2, Humans, Macrophages, Mice, Neoplasms, Oxidation-Reduction, Oxygen, Placental Lactogen, Signal Transduction, Spodoptera
Show Abstract · Added January 20, 2015
The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O(2). Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nm; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H(2)O(2). The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H(2)(18)O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.
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16 MeSH Terms
Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.
Ahonen MT, Diaconu I, Pesonen S, Kanerva A, Baumann M, Parviainen ST, Spiller B, Cerullo V, Hemminki A
(2010) PLoS One 5:
MeSH Terms: Adenoviridae, Animals, Buffers, Calcium Gluconate, Cell Line, Tumor, Cricetinae, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors, Humans, Mice, Mice, Inbred C57BL, Mice, Inbred ICR, Mice, Nude, Phosphates
Show Abstract · Added May 29, 2014
BACKGROUND - Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important.
METHODS/RESULTS - We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly.
CONCLUSION - In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.
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15 MeSH Terms
Electron microscopy of whole cells in liquid with nanometer resolution.
de Jonge N, Peckys DB, Kremers GJ, Piston DW
(2009) Proc Natl Acad Sci U S A 106: 2159-64
MeSH Terms: Animals, Buffers, COS Cells, Chlorocebus aethiops, Epidermal Growth Factor, Equipment Design, ErbB Receptors, Fibroblasts, Gold, Image Processing, Computer-Assisted, Metal Nanoparticles, Microscopy, Electron, Nanotechnology, Protein Binding, Silicon
Show Abstract · Added December 6, 2012
Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 micros were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 +/- 1 microm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.
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15 MeSH Terms
Analysis of protein composition using multidimensional chromatography and mass spectrometry.
Link AJ, Jennings JL, Washburn MP
(2004) Curr Protoc Protein Sci Chapter 23: Unit 23.1
MeSH Terms: Angiotensins, Buffers, Chromatography, Mass Spectrometry, Proteins, Software
Show Abstract · Added April 18, 2013
Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution.
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6 MeSH Terms
Stability of cytochrome P450 enzymes in human liver samples stored in different tissue preservation buffers.
Nishimura Y, Kurata N, Hanna IH, Guengerich FP, Yasuhara H
(2002) Res Commun Mol Pathol Pharmacol 111: 13-26
MeSH Terms: Buffers, Catalysis, Cytochrome P-450 Enzyme System, Enzyme Stability, Humans, Microsomes, Liver, Organ Preservation Solutions
Show Abstract · Added May 26, 2014
The effects of various tissue preservation buffers on cytochrome P450 (P450)-mediated activities of microsomes prepared from fresh surgical liver samples were examined. Two individual human liver samples were obtained, and three portions of each were preserved in one of three solutions: phosphate buffer, Krebs-Heinseleit solution, or University of Wisconsin solution. Microsomes were prepared within 24 h, 3 days, and 7 days after the resection of the samples. Marker P450 activities were measured. Liver microsomes prepared within 24 h displayed similar ethoxyresorufin O-deethylation, bufuralol 1'-hydroxylation (BF 1'-OH), and chlorzoxazone 6-hydroxylation activities in all preservation solutions, whereas S-mephenytoin 4'-hydroxylation and midazolam 1'-hydroxylation activities displayed some variation depending on the preservation buffer used. Most of the marker P450 activities were stable for 3 days after removal from surgical patients and declined at 7 days; however, a decline in BF-1'OH activity was observed even at day 3. These results suggest P450-specific activity depends on the method by which human liver samples are preserved. Moreover, the results of these studies establish the minimum tissue preservation criteria that, when met, qualify the drug disposition data generated from subcellular fractions derived from a particular surgical tissue sample.
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7 MeSH Terms
Effects of relative humidity and buffer additives on the contact printing of microarrays by quill pins.
McQuain MK, Seale K, Peek J, Levy S, Haselton FR
(2003) Anal Biochem 320: 281-91
MeSH Terms: Buffers, Fluorescence, Humidity, Oligonucleotide Array Sequence Analysis, Printing
Show Abstract · Added May 27, 2014
DNA microarrays printed with quill pins exhibit significant variation in probe DNA spots. Interspot variations and nonuniform distribution of probe within spots are major sources of experimental uncertainty in microarray analysis. To gain better insight into the sources of variation, we analyzed 450 consecutive depositions printed at relative humidities between 40 and 80% using three print buffers. Increasing relative humidity improved printing performance by delaying pin failure but did not reduce the variability in spot characteristics. Adding either betaine or dimethyl sulfoxide (DMSO) to the print buffer also improved quill pin performance. Least interspot variation was observed with the DMSO additive printed at 80% relative humidity, but this additive also resulted in the greatest intraspot variation. Least intraspot variation was observed with 1.5M betaine printed at 60% relative humidity, but these conditions produced microarrays with high interspot variability. Evaporation of printing solution from the quill reservoir appeared to be the primary cause of interspot and intraspot variations. Our studies indicate that relative humidity and printing solution additives reduce evaporation. Based on the spot variability requirements for a particular application, humidity and additives may be chosen to optimize either inter- or intraspot variability.
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5 MeSH Terms
Chemical stability of 2-arachidonylglycerol under biological conditions.
Rouzer CA, Ghebreselasie K, Marnett LJ
(2002) Chem Phys Lipids 119: 69-82
MeSH Terms: Arachidonic Acids, Buffers, Chromatography, High Pressure Liquid, Culture Media, Drug Stability, Endocannabinoids, Glycerides, Ligands, Oxidation-Reduction, Receptors, Cannabinoid, Receptors, Drug
Show Abstract · Added March 5, 2014
Recent evidence indicates that 2-arachidonylglycerol (2-AG) is a potent and specific ligand for the central and peripheral cannabinoid receptors. Therefore, the chemical stability of this molecule under biological conditions is of interest. A method for the isolation and detection of 2-AG using HPLC with evaporative light scattering detection is described. The method provides an extraction recovery from aqueous media of 78%, and a limit of detection of 60 ng on column. Incubation of 2-AG in culture medium or biological buffers indicated that it is stable to oxidation and ester hydrolysis for up to 6 h at 37 degrees C. However, gradual disappearance of the compound was noted due to adherence to glass and plastic surfaces. During incubation in RPMI culture medium, 2-AG rearranged to 1(3)-arachidonylglycerol (1(3)-AG) in a first order process with a half-life of 10 min in the absence of serum and 2.3 min in the presence of 10% fetal calf serum. Further studies indicated that the acyl migration reaction is base catalyzed (k(cat)=78,000/min M), and that the reaction is affected slightly by changes in buffer (Tris) concentration and not at all by changes in ionic strength. The results indicate that 2-AG is readily converted to 1(3)-AG under conditions commonly used to study receptor-ligand interactions, findings that have significant implications for the interpretation of relative ligand potency between the two isomers.
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11 MeSH Terms