105 research outputs found
A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein
We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes-based on the structure of ivacaftor-to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021)
Screening for Familial APP Mutations in Sporadic Cerebral Amyloid Angiopathy
Background
Advances in genetic technology have revealed that variation in the same gene can cause both rare familial and common sporadic forms of the same disease. Cerebral amyloid angiopathy (CAA), a common cause of symptomatic intracerebral hemorrhage (ICH) in the elderly, can also occur in families in an autosomal dominant pattern. The majority of affected families harbor mutations in the Beta amyloid Peptide (Aβ) coding region of the gene for amyloid precursor protein (APP) or have duplications of chromosomal segments containing APP.
Methodology/Principal Findings
A total of 58 subjects with a diagnosis of probable or definite CAA according to validated criteria were included in the present study. We sequenced the Aβ coding region of APP in 58 individuals and performed multiplex ligation-dependent probe amplification to determine APP gene dosage in 60. No patient harbored a known or novel APP mutation or gene duplication. The frequency of mutations investigated in the present study is estimated to range from 0% to 8% in individuals with probable CAA in the general population, based on the ascertained sample size.
Conclusions/Significance
We found no evidence that variants at loci associated with familial CAA play a role in sporadic CAA. Based on our findings, these rare highly-penetrant mutations are unlikely to be seen in sporadic CAA patients. Therefore, our results do not support systematic genetic screening of CAA patients who lack a strong family history of hemorrhage or dementia.National Institute of Neurological Disorders and Stroke (U.S.) (grant K23NS042695)American Heart AssociationAmerican Stroke Association (Bugher Foundation for Stroke Prevention Research
Mechanical exfoliation and layer number identification of single crystal monoclinic CrCl3
After the recent finding that CrI3, displays ferromagnetic order down to its monolayer, extensive studies have followed to pursue new two-dimensional (2D) magnetic materials. In this article, we report on the growth of single crystal CrCl3 in the layered monoclinic phase. The system after mechanical exfoliation exhibits stability in ambient air (the degradation occurs on a time scale at least four orders of magnitude longer than is observed for CrI3). By means of mechanical cleavage and atomic force microscopy (AFM) combined with optical identification, we demonstrate the systematic isolation of single and few layer flakes onto 270 nm and 285 nm SiO2/Si~(100) substrates with lateral size larger than graphene flakes isolated with the same method. The layer number identification has been carried with statistically significant data, quantifying the optical contrast as a function of the number of layers for up to six layers. Layer dependent optical contrast data have been fitted within the Fresnel equation formalism determining the real and imaginary part of the wavelength dependent refractive index of the material. A layer dependent (532 nm) micro-Raman study has been carried out down to two layers with no detectable spectral shifts as a function of the layer number and with respect to the bulk
Double-well magnetic trap for Bose-Einstein condensates
We present a magnetic trapping scheme for neutral atoms based on a hybrid of
Ioffe-Pritchard and Time-averaged Orbiting Potential traps. The resulting
double-well magnetic potential has readily controllable barrier height and well
separation. This offers a new tool for studying the behavior of Bose
condensates in double-well potentials, including atom interferometry and
Josephson tunneling. We formulate a description for the potential of this
magnetic trap and discuss practical issues such as loading with atoms,
evaporative cooling and manipulating the potential.Comment: 7 pages, 6 figures, Revtex
Screening for Familial APP Mutations in Sporadic Cerebral Amyloid Angiopathy
Background
Advances in genetic technology have revealed that variation in the same gene can cause both rare familial and common sporadic forms of the same disease. Cerebral amyloid angiopathy (CAA), a common cause of symptomatic intracerebral hemorrhage (ICH) in the elderly, can also occur in families in an autosomal dominant pattern. The majority of affected families harbor mutations in the Beta amyloid Peptide (Aβ) coding region of the gene for amyloid precursor protein (APP) or have duplications of chromosomal segments containing APP.
Methodology/Principal Findings
A total of 58 subjects with a diagnosis of probable or definite CAA according to validated criteria were included in the present study. We sequenced the Aβ coding region of APP in 58 individuals and performed multiplex ligation-dependent probe amplification to determine APP gene dosage in 60. No patient harbored a known or novel APP mutation or gene duplication. The frequency of mutations investigated in the present study is estimated to range from 0% to 8% in individuals with probable CAA in the general population, based on the ascertained sample size.
Conclusions/Significance
We found no evidence that variants at loci associated with familial CAA play a role in sporadic CAA. Based on our findings, these rare highly-penetrant mutations are unlikely to be seen in sporadic CAA patients. Therefore, our results do not support systematic genetic screening of CAA patients who lack a strong family history of hemorrhage or dementia.National Institute of Neurological Disorders and Stroke (U.S.) (grant K23NS042695)American Heart AssociationAmerican Stroke Association (Bugher Foundation for Stroke Prevention Research
Optimization of evaporative cooling towards a large number of Bose-Einstein condensed atoms
We study the optimization of evaporative cooling in trapped bosonic atoms on
the basis of quantum kinetic theory of a Bose gas. The optimized cooling
trajectory for Rb atoms indicates that the acceleration of evaporative
cooling around the transition point of Bose-Einstein condensation is very
effective against loss of trapped atoms caused by three-body recombination. The
number of condensed atoms is largely enhanced by the optimization, more than
two orders of magnitude in our present calculation using relevant experimental
parameters, as compared with the typical value given by the conventional
evaporative cooling where the frequency of radio-frequency magnetic field is
swept exponentially. In addition to this optimized cooling, it is also shown
that highly efficient evaporative cooling can be achieved by an initial
exponential and then a rapid linear sweep of frequency.Comment: 7 pages, REVTeX, 5 eps figures, Phys. Rev A in press (01 Feburuary
2003
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Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain
Background:
Protein tyrosine kinases are important regulators of cellular homeostasis with tightly
controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory
constraints on kinase activity, can promote malignant transformation, and appear to be a major
determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase
domain, for example, have recently been identified in patients who showed clinical responses
to EGFR kinase inhibitor therapy.
Methods and Findings:
Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR)
kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR
coding sequence in glioma tumor samples and cell lines. We identified novel missense
mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/
8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene
dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells.
Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR
kinase inhibitors.
Conclusions:
Our results suggest extracellular missense mutations as a novel mechanism for oncogenic
EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for
treatment of glioblastoma
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Exome and whole genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity
The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a five-year survival rate of 15%, identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole exome sequencing of 149 EAC tumors/normal pairs, 15 of which have also been subjected to whole genome sequencing. We identify a mutational signature defined by a high prevalence of A to C transversions at AA dinucleotides. Statistical analysis of exome data identified significantly mutated 26 genes. Of these genes, four (TP53, CDKN2A, SMAD4, and PIK3CA) have been previously implicated in EAC. The novel significantly mutated genes include chromatin modifying factors and candidate contributors: SPG20, TLR4, ELMO1, and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 reveal increased cellular invasion. Therefore, we suggest a new hypothesis about the potential activation of the RAC1 pathway to be a contributor to EAC tumorigenesis
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