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Potent DNA damage by polyhalogenated quinones and Hâ‚‚Oâ‚‚ via a metal-independent and Intercalation-enhanced oxidation mechanism
Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important center [dot]OH can be produced by polyhalogenated quinones and Hâ‚‚Oâ‚‚ independent of transition metal ions. However, it is not clear whether this unusual metal-independent center dot OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and Hâ‚‚Oâ‚‚ can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to center dot OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and Hâ‚‚Oâ‚‚ can induce potent DNA damage via a metal- independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicityKeywords: Halogenated quinones, Copper, Pentachlorophenol, Drinking water, Oxyradicals, Hydroxyl radicals, Hydrogen peroxide, Disinfection by products, Intracellular iron, Guanine moiet
Gadd45a promotes DNA demethylation through TDG
Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathwa
Spatial Segmentation of Mass Spectrometry Imaging Data by Combining Multivariate Clustering and Univariate Thresholding
Spatial segmentation partitions
mass spectrometry imaging (MSI) data into distinct regions providing a concise visualization
of the vast amount of data and identifying regions of interest (ROIs) for
downstream statistical analysis. Unsupervised approaches are particularly
attractive as they may be used to discover the underlying subpopulations present
in the high-dimensional MSI data without prior knowledge of the properties of
the sample. Herein, we introduce an unsupervised spatial segmentation approach,
which combines multivariate clustering and univariate thresholding to generate
comprehensive spatial segmentation maps of the MSI data. This approach combines
matrix factorization and manifold learning to enable high-quality image
segmentation without an extensive hyperparameter search. In parallel, some ion
images inadequately represented in the multivariate analysis are treated using
univariate thresholding to generate complementary spatial segments. The final
spatial segmentation map is assembled from segment candidates generated using both
techniques. We demonstrate the performance and robustness of this approach for
two MSI data sets of mouse uterine and kidney tissue sections acquired with
different spatial resolutions. The resulting segmentation maps are easy to
interpret and project onto the known anatomical regions of the tissue.</p
Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level
Quantitative live
cell mass spectrometry analysis at a subcellular
level requires the precisely controlled extraction of subpicoliter
volumes of material from the cell, sensitive analysis of the extracted
analytes, and their accurate quantification without prior separation.
In this study, we demonstrate that localized electroosmotic extraction
provides a direct path to addressing this challenge. Specifically,
we demonstrate quantitative mass spectrometry analysis of biomolecules
in picoliter volumes extracted from live cells. Electroosmotic extraction
was performed using two electrodes and a finely pulled nanopipette
with tip diameter of <1 μm containing a hydrophobic electrolyte
compatible with mass spectrometry analysis. The electroosmotic drag
was used to drive analytes out of the cell into the nanopipette. Analyte
molecules extracted both from solutions and cell samples were analyzed
using nanoelectrospray ionization (nanoESI) directly from the nanopipette
into a mass spectrometer. More than 50 metabolites including sugars
and flavonoids were detected in positive mode in 2−5 pL volumes
of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential
extraction of a known volume of the aqueous solution containing glucose-<i>d</i><sub>2</sub> standard of known concentration. We found
that the ratio of the signal of glucose to glucose-<i>d</i><sub>2</sub> increased linearly with glucose concentration. This
observation indicates that the approach developed in this study enables
quantitative analysis of small volumes of metabolites extracted from
cells. Furthermore, we observed efficient separation of hydrophilic
and hydrophobic analytes through partitioning into the aqueous and
hydrophobic electrolyte phase, respectively, which provides additional
important information on the molecular properties of extracted metabolites
Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level
Quantitative live
cell mass spectrometry analysis at a subcellular
level requires the precisely controlled extraction of subpicoliter
volumes of material from the cell, sensitive analysis of the extracted
analytes, and their accurate quantification without prior separation.
In this study, we demonstrate that localized electroosmotic extraction
provides a direct path to addressing this challenge. Specifically,
we demonstrate quantitative mass spectrometry analysis of biomolecules
in picoliter volumes extracted from live cells. Electroosmotic extraction
was performed using two electrodes and a finely pulled nanopipette
with tip diameter of <1 μm containing a hydrophobic electrolyte
compatible with mass spectrometry analysis. The electroosmotic drag
was used to drive analytes out of the cell into the nanopipette. Analyte
molecules extracted both from solutions and cell samples were analyzed
using nanoelectrospray ionization (nanoESI) directly from the nanopipette
into a mass spectrometer. More than 50 metabolites including sugars
and flavonoids were detected in positive mode in 2−5 pL volumes
of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential
extraction of a known volume of the aqueous solution containing glucose-<i>d</i><sub>2</sub> standard of known concentration. We found
that the ratio of the signal of glucose to glucose-<i>d</i><sub>2</sub> increased linearly with glucose concentration. This
observation indicates that the approach developed in this study enables
quantitative analysis of small volumes of metabolites extracted from
cells. Furthermore, we observed efficient separation of hydrophilic
and hydrophobic analytes through partitioning into the aqueous and
hydrophobic electrolyte phase, respectively, which provides additional
important information on the molecular properties of extracted metabolites
Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level
Quantitative live
cell mass spectrometry analysis at a subcellular
level requires the precisely controlled extraction of subpicoliter
volumes of material from the cell, sensitive analysis of the extracted
analytes, and their accurate quantification without prior separation.
In this study, we demonstrate that localized electroosmotic extraction
provides a direct path to addressing this challenge. Specifically,
we demonstrate quantitative mass spectrometry analysis of biomolecules
in picoliter volumes extracted from live cells. Electroosmotic extraction
was performed using two electrodes and a finely pulled nanopipette
with tip diameter of <1 μm containing a hydrophobic electrolyte
compatible with mass spectrometry analysis. The electroosmotic drag
was used to drive analytes out of the cell into the nanopipette. Analyte
molecules extracted both from solutions and cell samples were analyzed
using nanoelectrospray ionization (nanoESI) directly from the nanopipette
into a mass spectrometer. More than 50 metabolites including sugars
and flavonoids were detected in positive mode in 2−5 pL volumes
of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential
extraction of a known volume of the aqueous solution containing glucose-<i>d</i><sub>2</sub> standard of known concentration. We found
that the ratio of the signal of glucose to glucose-<i>d</i><sub>2</sub> increased linearly with glucose concentration. This
observation indicates that the approach developed in this study enables
quantitative analysis of small volumes of metabolites extracted from
cells. Furthermore, we observed efficient separation of hydrophilic
and hydrophobic analytes through partitioning into the aqueous and
hydrophobic electrolyte phase, respectively, which provides additional
important information on the molecular properties of extracted metabolites
Nickel(II) Inhibits Tet-Mediated 5‑Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II)
Ten-eleven
translocation (Tet) family proteins are FeÂ(II)- and
2-oxoglutarate-dependent dioxygenases that regulate the dynamics of
DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine
(5mC). To exert physiologically important functions, redox-active
iron chelated in the catalytic center of Tet proteins directly involves
the oxidation of the multiple substrates. To understand the function
and interaction network of Tet dioxygenases, it is interesting to
obtain high affinity and a specific inhibitor. Surprisingly, here
we found that natural NiÂ(II) ion can bind to the FeÂ(II)-chelating
motif (HXD) with an affinity of 7.5-fold as high as FeÂ(II). Consistently,
we further found that NiÂ(II) ion can displace the cofactor FeÂ(II)
of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity
with an estimated IC<sub>50</sub> of 1.2 μM. Essentially, NiÂ(II)
can be used as a high affinity and selective inhibitor to explore
the function and dynamics of Tet proteins
Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing
A capillary monolithic bioreactor of snake venom phosphodiesterase
(SVP) was constructed to generate different single-nucleotide mass
ladders of oligodeoxynucleotides for mass spectrometry (MS)-based
sequencing by immobilization. The immobilization of SVP in the porous
silica monolith significantly enhances its stability for prolonged
and repeated applications. The constructed capillary bioreactor has
the advantages of handling (sub)Âmicroliter DNA samples and having
good permeability. Benefiting from its good permeability, DNA solutions
can be directly injected into the sequential digestion bioreactor
simply by hand pushing or a low-pressure microinjection pump. Moreover,
the immobilization of SVP facilitates the elimination or repression
of the metal adducts of oligodeoxynucleotides, improving the analytical
performance of MS sequencing. By the application of capillary bioreactor
of immobilized SVP, the sequence-specific modification of single-stranded
oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr)
was identified, demonstrating its promising applications in identification
of sequence-specific damage, which may further our understanding of
DNA damage caused mutagenesis
Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry Imaging of Drift Time-Separated Ions
Simultaneous spatial localization and structural characterization of
molecules in complex biological samples currently represents an analytical
challenge for mass spectrometry imaging (MSI) techniques. In this study, we
describe a novel experimental platform, which substantially expands the
capabilities and enhances the depth of chemical information obtained in high
spatial resolution MSI experiments performed using nanospray desorption
electrospray ionization (nano-DESI). Specifically, we designed and constructed
a portable nano-DESI MSI platform and coupled it with a drift tube ion mobility
spectrometer-mass spectrometer (IM-MS). Separation of biomolecules observed in
MSI experiments based on their drift times provides unique molecular
descriptors necessary for their identification by comparison with databases. Furthermore,
it enables isomer-specific imaging, which is particularly important for
unraveling the complexity of biological systems. Imaging of day 4 pregnant
mouse uterine sections using the newly developed nano-DESI-IM-MSI system demonstrates
rapid isobaric and isomeric separation
and reduced chemical noise in MSI experiments. A direct comparison of the
performance of the new nano-DESI-MSI platform operated in the MS mode with the
more established nano-DESI-Orbitrap platform indicates a comparable performance
of these two systems. A spatial resolution of better than ~16 µm and similar molecular
coverage was obtained using both platforms.
The structural information provided by the ion mobility separation
expands the molecular specificity of high-resolution MSI necessary for the
detailed understanding of biological systems