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  1. Article ; Online: Differential scanning calorimetry of gliomas: a new tool in brain cancer diagnostics?

    Chagovetz, Alexis A / Quinn, Colette / Damarse, Neil / Hansen, Lee D / Chagovetz, Alexander M / Jensen, Randy L

    Neurosurgery

    2013  Volume 73, Issue 2, Page(s) 289–95; discussion 295

    Abstract: Background: Thermal stability signatures of complex molecular interactions in biological fluids can be measured using differential scanning calorimetry (DSC). Evaluating the thermal stability of plasma proteomes offers a method of producing a disease- ... ...

    Abstract Background: Thermal stability signatures of complex molecular interactions in biological fluids can be measured using differential scanning calorimetry (DSC). Evaluating the thermal stability of plasma proteomes offers a method of producing a disease-specific "signature" (thermogram) in neoplastic and autoimmune diseases.
    Objective: The authors describe the use of DSC with human brain tumor tissue to create unique thermograms for correlation with histological tumor classification.
    Methods: Primary brain tumors were classified according to the World Health Organization classification. Tumor samples were digested and assayed by a DSC calorimeter. Experimental thermograms were background subtracted and normalized to the total area of transitions to exclude concentration effects. The resulting thermograms were analyzed by applying 2-state, scaled, Gaussian distributions.
    Results: Differences in glioma-specific signatures are described by using calculated parameters at transitions that are characterized, in the equilibrium approximation, by a melting temperature (Tm), an apparent enthalpy change (ΔH), and a scaling factor related to the relative abundance of the materials denatured in the transition (Aw). Thermogram signatures of glioblastoma multiforme and low-grade astrocytomas were differentiated by calculated values of Aw3 and Tm4, those of glioblastoma multiforme and oligodendrogliomas were differentiated by Aw2, ΔH2, ΔH4, and Tm4, and those of low-grade astrocytomas and oligodendroglioma were differentiated by Aw4.
    Conclusion: Our preliminary results suggest that solid brain tumors exhibit specific thermogram profiles that are distinguishable among glioma grades. We anticipate that our results will form the conceptual base of a novel diagnostic assay based on tissue thermograms as a complement to currently used histological analysis.
    MeSH term(s) Adult ; Aged ; Brain Neoplasms/classification ; Brain Neoplasms/pathology ; Calorimetry, Differential Scanning/methods ; Female ; Glioma/classification ; Glioma/pathology ; Humans ; Male ; Middle Aged ; Neoplasm Grading ; Young Adult
    Language English
    Publishing date 2013-08
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 135446-2
    ISSN 1524-4040 ; 0148-396X
    ISSN (online) 1524-4040
    ISSN 0148-396X
    DOI 10.1227/01.neu.0000430296.23799.cd
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Real-time DNA microarrays: reality check.

    Chagovetz, Alexander / Blair, Steve

    Biochemical Society transactions

    2009  Volume 37, Issue Pt 2, Page(s) 471–475

    Abstract: DNA microarrays are plagued with inconsistent quantifications and false-positive results. Using established mechanisms of surface reactions, we argue that these problems are inherent to the current technology. In particular, the problem of multiplex non- ... ...

    Abstract DNA microarrays are plagued with inconsistent quantifications and false-positive results. Using established mechanisms of surface reactions, we argue that these problems are inherent to the current technology. In particular, the problem of multiplex non-equilibrium reactions cannot be resolved within the framework of the existing paradigm. We discuss the advantages and limitations of changing the paradigm to real-time data acquisition similar to real-time PCR methodology. Our analysis suggests that the fundamental problem of multiplex reactions is not resolved by the real-time approach itself. However, by introducing new detection chemistries and analysis approaches, it is possible to extract target-specific quantitative information from real-time microarray data. The possible scope of applications for real-time microarrays is discussed.
    MeSH term(s) Algorithms ; Computer Simulation ; DNA/genetics ; Genomics ; Oligonucleotide Array Sequence Analysis/instrumentation ; Oligonucleotide Array Sequence Analysis/methods
    Chemical Substances DNA (9007-49-2)
    Language English
    Publishing date 2009-03-17
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 184237-7
    ISSN 1470-8752 ; 0300-5127
    ISSN (online) 1470-8752
    ISSN 0300-5127
    DOI 10.1042/BST0370471
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  3. Article ; Online: Preliminary use of differential scanning calorimetry of cerebrospinal fluid for the diagnosis of glioblastoma multiforme.

    Chagovetz, Alexis A / Jensen, Randy L / Recht, Larry / Glantz, Michael / Chagovetz, Alexander M

    Journal of neuro-oncology

    2011  Volume 105, Issue 3, Page(s) 499–506

    Abstract: Thermal stability signatures of complex molecule interaction in biological fluids can be measured using a new approach called differential scanning calorimetry (DSC). The thermal stability of plasma proteome has been described previously as a method of ... ...

    Abstract Thermal stability signatures of complex molecule interaction in biological fluids can be measured using a new approach called differential scanning calorimetry (DSC). The thermal stability of plasma proteome has been described previously as a method of producing a disease-specific "signature," termed thermogram, in several neoplastic and autoimmune diseases. We describe the preliminary use of DSC performed on cerebrospinal fluid (CSF) as a diagnostic tool for the identification of patients with glioblastoma multiforme (GBM). Samples of CSF from nine patients with confirmed GBM were evaluated using DSC, and the thermogram signatures evaluated. These thermograms were compared with thermograms of CSF taken from patients with non-neoplastic conditions such as head trauma, hydrocephalus, or CSF leak. Further analysis was also performed on CSF from patients who had non-GBM neoplastic conditions such as carcinomatosis meningitis or central nervous system lymphoma or leukemia. The DSC thermograms of CSF of the patients with GBM were significantly different when compared with other neoplastic and non-neoplastic cases. The melting temperature of the major transition was shifted by 5°C, which makes it easily distinguishable from control cases. Our results are very preliminary, but it appears that the DSC of CSF has potential utility in diagnostics and monitoring disease progression in GBM patients.
    MeSH term(s) Brain Neoplasms/cerebrospinal fluid ; Brain Neoplasms/diagnosis ; Calorimetry, Differential Scanning ; Cerebrospinal Fluid/chemistry ; Glioblastoma/cerebrospinal fluid ; Glioblastoma/diagnosis ; Humans
    Language English
    Publishing date 2011-07-01
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 604875-4
    ISSN 1573-7373 ; 0167-594X
    ISSN (online) 1573-7373
    ISSN 0167-594X
    DOI 10.1007/s11060-011-0630-5
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Competitive displacement: a sensitive and selective method for the detection of unlabeled molecules.

    Bishop, Justin / Chagovetz, Alexander M / Blair, Steve

    Optics express

    2009  Volume 15, Issue 8, Page(s) 4390–4397

    Abstract: We propose a new method for molecular detection that retains the sensitivity of fluorescence, but without requiring fluorescence labeling of the sample. The method works by spiking the sample solution with one or more labeled molecular species of known ... ...

    Abstract We propose a new method for molecular detection that retains the sensitivity of fluorescence, but without requiring fluorescence labeling of the sample. The method works by spiking the sample solution with one or more labeled molecular species of known concentration. With proper choice of these "competitor" species, their binding kinetics can be used to quantitatively determine the concentration of unlabeled target species. This method can be applied to any fluorescence transduction mechanism that allows real-time signal acquisition, and represents an advance in mitigating certain sample processing steps. We demonstrate the method for the detection of a DNA sequence containing a single-nucleotide polymorphism (SNP).
    Language English
    Publishing date 2009-08-08
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1491859-6
    ISSN 1094-4087 ; 1094-4087
    ISSN (online) 1094-4087
    ISSN 1094-4087
    DOI 10.1364/oe.15.004390
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article: Microarray temperature optimization using hybridization kinetics.

    Blair, Steve / Williams, Layne / Bishop, Justin / Chagovetz, Alexander

    Methods in molecular biology (Clifton, N.J.)

    2009  Volume 529, Page(s) 171–196

    Abstract: In any microarray hybridization experiment, there are contributions at each probe spot due to the match and numerous mismatch target species (i.e., cross-hybridizations). One goal of temperature optimization is to minimize the contribution of mismatch ... ...

    Abstract In any microarray hybridization experiment, there are contributions at each probe spot due to the match and numerous mismatch target species (i.e., cross-hybridizations). One goal of temperature optimization is to minimize the contribution of mismatch species; however, achieving this goal may come at the expense of obtaining equilibrium reaction conditions. We employ two-component thermodynamic and kinetic models to study the trade-offs involved in temperature optimization. These models show that the maximum selectivity is achieved at equilibrium, but that the mismatch species controls the time to equilibrium via the competitive displacement mechanism. Also, selectivity is improved at lower temperatures. However, the time to equilibrium is also extended, so that greater selectivity cannot be achieved in practice. We also employ a two-color real-time microarray reader to experimentally demonstrate these effects by independently monitoring the match and mismatch species during multiplex hybridization. The only universal criterion that can be employed is to optimize temperature based upon attaining equilibrium reaction conditions. This temperature varies from one probe to another, but can be determined empirically using standard microarray experimentation methods.
    MeSH term(s) Fluorescence ; Kinetics ; Oligonucleotide Array Sequence Analysis/instrumentation ; Oligonucleotide Array Sequence Analysis/methods ; Solutions ; Temperature ; Thermodynamics ; Time Factors
    Chemical Substances Solutions
    Language English
    Publishing date 2009
    Publishing country United States
    Document type Journal Article
    ISSN 1064-3745
    ISSN 1064-3745
    DOI 10.1007/978-1-59745-538-1_12
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  6. Article ; Online: The paradox of multiplex DNA melting on a surface.

    Williams, Layne / Blair, Steve / Chagovetz, Alexander / Fish, Daniel J / Benight, Albert S

    Analytical biochemistry

    2010  Volume 409, Issue 1, Page(s) 150–152

    Abstract: Under equilibrium conditions, there are two regimes of target capture on a surface--target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a ...

    Abstract Under equilibrium conditions, there are two regimes of target capture on a surface--target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a reverse displacement mechanism of the low affinity species. The melting curve cannot be used in analytical methods to resolve heteroduplexes; only with the simplex system can proper thermodynamics be obtained.
    MeSH term(s) Base Sequence ; DNA/chemistry ; Nucleic Acid Denaturation ; Thermodynamics ; Transition Temperature
    Chemical Substances DNA (9007-49-2)
    Language English
    Publishing date 2010-10-30
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2010.09.024
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    Zs.A 389: Show issues Location:
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  7. Article: The paradox of multiplex DNA melting on a surface

    Williams, Layne / Blair, Steve / Chagovetz, Alexander / Fish, Daniel J / Benight, Albert S

    Analytical biochemistry. 2011 Feb. 1, v. 409, no. 1

    2011  

    Abstract: Under equilibrium conditions, there are two regimes of target capture on a surface – target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to ... ...

    Abstract Under equilibrium conditions, there are two regimes of target capture on a surface – target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a reverse displacement mechanism of the low affinity species. The melting curve cannot be used in analytical methods to resolve heteroduplexes; only with the simplex system can proper thermodynamics be obtained.
    Keywords DNA ; analytical methods ; dissociation ; melting ; thermodynamics
    Language English
    Dates of publication 2011-0201
    Size p. 150-152.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2010.09.024
    Database NAL-Catalogue (AGRICOLA)

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  8. Article ; Online: Direct DNA methylation profiling using methyl binding domain proteins.

    Yu, Yinni / Blair, Steve / Gillespie, David / Jensen, Randy / Myszka, David / Badran, Ahmed H / Ghosh, Indraneel / Chagovetz, Alexander

    Analytical chemistry

    2010  Volume 82, Issue 12, Page(s) 5012–5019

    Abstract: Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer ... ...

    Abstract Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the K(D) for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The K(D) values for nonsymmetrical methylation motifs were consistently greater (>2 muM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.
    MeSH term(s) Base Sequence ; CpG Islands ; DNA/analysis ; DNA Methylation ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; Oligonucleotide Array Sequence Analysis/methods ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Sensitivity and Specificity ; Surface Plasmon Resonance/methods
    Chemical Substances DNA-Binding Proteins ; Recombinant Proteins ; Green Fluorescent Proteins (147336-22-9) ; DNA (9007-49-2)
    Language English
    Publishing date 2010-05-21
    Publishing country United States
    Document type Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/ac1010316
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