ABSTRACT
Traditional matrices for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) are usually crystalline small molecules. The heterogeneous co-crystallization of the analyte and the matrix creates a sweet spot effect and reduces point-to-point reproducibility. In this study, an amorphous poly-N-vinylcarbazole polymer (PVK) was studied as a novel matrix for MALDI-TOF MS to detect various low molecular weight compounds (LMWCs) in the negative ion mode. The PVK achieved excellent matrix action and showed high sensitivity, good salt tolerance, and reproducibility. These results significantly broaden the design rules for new and efficient polymeric MALDI matrices.
ABSTRACT
The efficient capture of multi-pollutant residues in food is vital for food safety monitoring. In this study, in-situ-fabricated magnetic MIL-53(Al) metal organic frameworks (MOFs), with good magnetic responsiveness, were synthesized and applied for the magnetic solid-phase extraction (MSPE) of chloramphenicol, bisphenol A, estradiol, and diethylstilbestrol. Terephthalic acid (H2BDC) organic ligands were pre-coupled on the surface of amino-Fe3O4 composites (H2BDC@Fe3O4). Fe3O4@MIL-53(Al) MOF was fabricated by in-situ hydrothermal polymerization of H2BDC, Al (NO3)3, and H2BDC@Fe3O4. This approach highly increased the stability of the material. The magnetic Fe3O4@MIL-53(Al) MOF-based MSPE was combined with high-performance liquid chromatography-photo diode array detection, to establish a novel sensitive method for analyzing multi-pollutant residues in milk. This method showed good linear correlations, in the range of 0.05-5.00 µg/mL, with good reproducibility. The limit of detection was 0.004-0.108 µg/mL. The presented method was verified using a milk sample, spiked with four pollutants, which enabled high-throughput detection and the accuracies of 88.17-107.58% confirmed its applicability, in real sample analysis.
Subject(s)
Environmental Pollutants , Metal-Organic Frameworks , Animals , Chromatography, High Pressure Liquid/methods , Environmental Pollutants/analysis , Limit of Detection , Magnetic Phenomena , Metal-Organic Frameworks/chemistry , Milk/chemistry , Reproducibility of Results , Solid Phase Extraction/methodsABSTRACT
A low-field nuclear magnetic resonance (LF-NMR) DNA-hydrogel (LNDH) nanoprobe was designed for bisphenol A (BPA) determination. It consists of Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) and a DNA-hydrogel technology. Fe3O4 SPIONs were encapsulated in the DNA-hydrogel to form an aggregated state. After adding BPA, the gel system transformed into a sol gel due to the target-aptamer specific binding. The coated gathered particles dispersed and thus, the relaxation time T2 declined. The LNDH nanoprobe was developed to realize a simple, sensitive, and effective BPA determination method without repeated magnetic separation steps. Under the optimal experimental conditions, the determination range of the LNDH biosensor was 10-2~102 ng mL-1 and the limit of determination was 0.07 ng mL-1. The LNDH nanoprobe was applied to two kinds of water samples (tap water and bottled water). The recovery ranged from 87.85 to approximately 97.87%. This strategy offered a new method to detect BPA by LF-NMR. It is also expected to be applicable in related fields of food safety determination, environmental monitoring, and clinical diagnosis. Graphical abstract Schematic presentation of LNDH biosensor. Acrydite-modified ssDNA was copolymerized with acrylamide to form linear conjugates PS-A/B, adding aptamer and SPIONs to form DNA-hydrogel. When aptamer captured the target, the hydrogel was destroyed to disperse the coated SPIONs. T2 relaxation time declined.
Subject(s)
Benzhydryl Compounds/analysis , DNA, Single-Stranded/chemistry , Drinking Water/chemistry , Hydrogels/chemistry , Magnetite Nanoparticles/chemistry , Phenols/analysis , Water Pollutants, Chemical/analysis , Aptamers, Nucleotide/chemistry , Benzhydryl Compounds/chemistry , Biosensing Techniques , Limit of Detection , Magnetic Resonance Spectroscopy , Phenols/chemistryABSTRACT
Based on superparamagnetic nanoparticles, a responsive polyacrylamide hydrogel self-assembled by nucleic acid hairpin hybridization chain reaction was designed, and a universal low field nuclear magnetic resonance sensing platform was successfully constructed. As the target was gradually added, the hydrogel coating on the surface of the magnetic nanoparticle was opened layer by layer through binding with the aptamer, which specifically bonded thereto, causing different degrees of exposure of the magnetic nanoparticle, resulting in changes of low field nuclear magnetic resonance signals. This method was originally applied to the rapid detection of adenosine triphosphate (ATP), and the versatility of the method was verified using polychlorinated biphenyl 77 (PCB77). This method had the advantage of being fast, convenient, and low cost, and it can be easily operated with high repeatability. This universal method can detect a variety of targets by replacing aptamers and may be useful in controlling food quality and for rapidly detecting cancer cells in vitro.
Subject(s)
Acrylic Resins/chemistry , Adenosine Triphosphate/blood , Hydrogels/chemistry , Magnetite Nanoparticles/chemistry , Polychlorinated Biphenyls/analysis , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Sequence , Biosensing Techniques/methods , Cattle , DNA/chemistry , DNA/genetics , Drinking Water/analysis , Inverted Repeat Sequences , Limit of Detection , Magnetic Resonance Spectroscopy/methods , Nucleic Acid Hybridization , Water Pollutants, Chemical/analysisABSTRACT
Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.
Subject(s)
DNA Repair/physiology , Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Rad51 Recombinase/genetics , Animals , Blotting, Western , Cell Line, Tumor , Fusion Proteins, bcr-abl/metabolism , Humans , Mice , Polymerase Chain Reaction , Rad51 Recombinase/metabolism , Transfection , Tyrosine/metabolismABSTRACT
In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.
Subject(s)
Homeodomain Proteins/physiology , Rhombencephalon/embryology , Somatosensory Cortex/anatomy & histology , Trigeminal Nerve/embryology , Vibrissae/innervation , Afferent Pathways , Animals , Axons/ultrastructure , Face/innervation , Homeodomain Proteins/genetics , Lip/innervation , Mandible/embryology , Mandible/innervation , Mice , Mice, Transgenic , Mutation , Neurons, Afferent/cytology , Receptor, EphA4/metabolism , Receptor, EphA7/metabolism , Rhombencephalon/cytology , Rhombencephalon/metabolism , Somatosensory Cortex/embryology , Thalamus/embryology , Thalamus/metabolism , Trigeminal Ganglion/embryology , Trigeminal Ganglion/metabolism , Trigeminal Nerve/physiology , Ventral Thalamic Nuclei/embryologyABSTRACT
OBJECTIVE: BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias. We show that a signaling protein, phosphatidylinositol-3 kinase (PI-3k), is essential for growth of CML cells, but not of normal hematopoietic cells, and that p85alpha subunit of PI-3k co-immunoprecipitates with BCR/ABL. Therefore, we made an attempt to better characterize p85alpha-BCR/ABL interactions. MATERIALS AND METHODS: The mutants of p85alpha-SH3 domain were generated by in vitro site-directed mutagenesis system. Protein lysates were obtained from p210BCR/ABL-transformed murine 32Dcl3 myeloid cells, and in vitro transcription/translation was used to produce BCR/ABL protein. Pull-down and Western analyses were performed to detect the interaction between BCR/ABL and p85alpha-SH3. BCR/ABL-transformed 32Dcl3 cells were infected with internal ribosome entry site-green fluorescent protein retroviral construct encoding p85alpha-SH3 mutants to assess their biological effects. RESULTS: We show here that the SH3 domain of p85alpha (p85alpha-SH3) pulls down the p210BCR/ABL kinase from hematopoietic cell lysates. The interaction between p85alpha-SH3 and BCR/ABL may be intermediated by proteins such as c-Cbl, Shc, Grb2, and/or Gab2. Mutations in the p85alpha-SH3 region responsible for proline-rich motif binding either abrogate or enhance these interactions. These mutants exert a modest inhibitory effect on growth factor-independent proliferation of BCR/ABL-positive 32Dcl3 cells. CONCLUSIONS: Based on this information we speculate on the capability of p85alpha-SH3 to interact with the protein network of BCR/ABL oncoprotein.
Subject(s)
Philadelphia Chromosome , Phosphatidylinositol 3-Kinases/metabolism , Protein Subunits/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , src Homology Domains/genetics , Amino Acid Substitution , Animals , Cell Line, Tumor , Fusion Proteins, bcr-abl , Hematopoietic Stem Cells/metabolism , Humans , Immunoprecipitation/methods , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases/genetics , Point Mutation/genetics , Protein Binding , Protein Subunits/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/geneticsABSTRACT
Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.
Subject(s)
Homeodomain Proteins/physiology , Neural Crest/embryology , Skull/embryology , Animals , Branchial Region/embryology , Ear, External/abnormalities , Ear, External/embryology , Ear, Middle/embryology , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Time FactorsABSTRACT
The p85alpha subunit of phosphatidylinositol 3-kinase (PI-3k) forms a complex with a protein network associated with oncogenic fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFbetaR, and NPM/ALK, resulting in constitutive activation of the p110 catalytic subunit of PI-3k. Introduction of point mutations in the N-terminal and C-terminal SH2 domain and SH3 domain of p85alpha, which disrupt their ability to bind phosphotyrosine and proline-rich motifs, respectively, abrogated their interaction with the BCR/ABL protein network. The p85alpha mutant protein (p85mut) bearing these mutations was unable to interact with BCR/ABL and other FTKs, while its binding to the p110alpha catalytic subunit of PI-3k was intact. In addition, binding of Shc, c-Cbl, and Gab2, but not Crk-L, to p85mut was abrogated. p85mut diminished BCR/ABL-dependent activation of PI-3k and Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of the hematopoietic cell line and murine bone marrow cells. Interestingly, the addition of interleukin-3 (IL-3) rescued BCR/ABL-transformed cells from the inhibitory effect of p85mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85mut survived longer than the animals inoculated with BCR/ABL-transformed counterparts. In conclusion, we have identified the domains of p85alpha responsible for the interaction with the FTK protein network and transduction of leukemogenic signaling.
Subject(s)
Fusion Proteins, bcr-abl/metabolism , Leukemia/metabolism , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cattle , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Humans , Leukemia/genetics , Mice , Mice, SCID , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphotyrosine/metabolism , Protein Interaction Mapping , src Homology DomainsABSTRACT
Recent studies indicated that retention of selectable marker cassettes in targeted Hox loci may cause unexpected phenotypes in mutant mice, due to neighborhood effects. However, the molecular mechanisms have been poorly investigated. Here, we analysed the effects of the targeted insertion of a PGK-neo cassette in the 3' untranslated region of Hoxa2. Even at this 3' position, the insertion resulted in homozygous mutants that unexpectedly did not survive beyond 3 weeks of age. Molecular analysis of the targeted allele revealed a selective "knockdown" of Hoxa2 expression in rhombomere 2 and associated patterning abnormalities. Moreover, Hoxa1 was ectopically expressed in the hindbrain and branchial arches of mutant embryos. Of interest, we demonstrated that the ectopic expression was due to the generation of neo-Hoxa1 fusion transcripts, resulting from aberrant alternative splicing. These defects could be rescued after removal of the PGK-neo cassette by Flp-mediated recombination. These results underscore the complexity of transcriptional regulation at Hox loci and provide insights into the in vivo regulation of Hoxa2 segmental expression. They also provide a molecular basis for the interpretation of unexpected Hox knockout phenotypes in which the targeted selectable marker is retained in the locus.