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Molecular Neurobiology Aug 2021Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular amyloid beta peptides and neurofibrillary tangles consisted of... (Review)
Review
Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular amyloid beta peptides and neurofibrillary tangles consisted of intracellular hyperphosphorylated Tau in the hippocampus and cerebral cortex. Most of the mutations in key genes that code for amyloid precursor protein can lead to significant accumulation of these peptides in the brain and cause Alzheimer's disease. Moreover, some point mutations in amyloid precursor protein can cause familial Alzheimer's disease, such as Swedish mutation (KM670/671NL) and A673V mutation. However, recent studies have found that the A673T mutation in amyloid precursor protein gene can protect against Alzheimer's disease, even if it is located next to the Swedish mutation (KM670/671NL) and at the same site as A673V mutation, which are pathogenic. It makes us curious about the protective A673T mutation. Here, we summarize the most recent insights of A673T mutation, focus on their roles in protective mechanisms against Alzheimer's disease, and discuss their involvement in future treatment.
Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Female; Humans; Male; Middle Aged; Point Mutation; Protein Structure, Secondary
PubMed: 33914267
DOI: 10.1007/s12035-021-02385-y -
Methods in Molecular Biology (Clifton,... 2024Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The... (Review)
Review
Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines.
Topics: Animals; DNA, Single-Stranded; Zebrafish; Gene Editing; Mutation; Point Mutation; Nucleotides
PubMed: 37668920
DOI: 10.1007/978-1-0716-3401-1_19 -
Analytical and Bioanalytical Chemistry Mar 2023Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to... (Review)
Review
Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.
Topics: Humans; Point Mutation; Mutation; Neoplasms; DNA; Biosensing Techniques; High-Throughput Nucleotide Sequencing; Genetic Predisposition to Disease
PubMed: 36289102
DOI: 10.1007/s00216-022-04388-7 -
Cellular and Molecular Neurobiology Oct 2023The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal...
The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb1 mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.
Topics: Animals; Mice; Hydrocephalus; Mutation; Point Mutation; Signal Transduction; Transcription Factor AP-1
PubMed: 37219662
DOI: 10.1007/s10571-023-01361-5 -
Journal of Agricultural and Food... Feb 2023Cyclobutrifluram is a novel succinate dehydrogenase inhibitor (SDHI) developed by Syngenta and helps to inhibit . Here, the potential for cyclobutrifluram resistance in...
Cyclobutrifluram is a novel succinate dehydrogenase inhibitor (SDHI) developed by Syngenta and helps to inhibit . Here, the potential for cyclobutrifluram resistance in and the resistance mechanism involved were evaluated. Baseline sensitivity of to cyclobutrifluram was determined with a mean EC value of 0.0248 μg/mL. Fungicide adaption generated five resistant mutants, which possess a comparable or a slightly impaired fitness compared to corresponding parental isolates. This indicates that the resistance risk of to cyclobutrifluram might be moderate. Cyclobutrifluram-resistant isolates also demonstrated resistance to pydiflumetofen but sensitivity to carbendazim, phenamacril, tebuconazole, fludioxonil, or pyraclostrobin. Additionally, point mutations H248Y in FpSdhB and A83V or R86K in FpSdhC were found in cyclobutrifluram-resistant mutants. Molecular docking and overexpression transformation assay revealed that FpSdhB and FpSdhC or FpSdhC confer the resistance of to cyclobutrifluram.
Topics: Point Mutation; Drug Resistance, Fungal; Succinate Dehydrogenase; Molecular Docking Simulation; Fungicides, Industrial; Fusarium; Plant Diseases
PubMed: 36657474
DOI: 10.1021/acs.jafc.2c08022 -
Current Protocols in Chemical Biology Sep 2019Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the...
Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the cassette to its intended location. These homology arms can be easily introduced as 5' primer overhangs during the PCR reaction. The flexibility to choose almost any pair of homology arms enables the precise modification of virtually any DNA for purposes of sequence deletion, replacement, insertion, or point mutation. Recombineering often offers significant advantages relative to previous homologous recombination methods that require the construction of cassettes with large homology arms, and relative to traditional cloning methods that become intractable for large plasmids or DNA sequences. However, the tremendous number of variables, options, and pitfalls that can be encountered when designing and performing a recombineering protocol for the first time introduce barriers that can make recombineering a challenging technique for new users to adopt. This article focuses on three recombineering protocols we have found to be particularly robust, providing a detailed guide for choosing the simplest recombineering method for a given application and for performing and troubleshooting experiments. © 2019 by John Wiley & Sons, Inc.
Topics: DNA; Escherichia coli; Gene Deletion; Genetic Engineering; Mutagenesis, Insertional; Plasmids; Point Mutation; Polymerase Chain Reaction; Research Design
PubMed: 31483098
DOI: 10.1002/cpch.70 -
Cell Mar 2020Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene that creates hemoglobin S (HbS). Upon deoxygenation, HbS forms long polymers that distort...
Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene that creates hemoglobin S (HbS). Upon deoxygenation, HbS forms long polymers that distort the shape of red blood cells, causing hemolysis and vaso-occlusion. Voxelotor inhibits HbS polymerization, the root cause of SCD complications. To view this Bench to Bedside, open or download the PDF.
Topics: Anemia, Sickle Cell; Benzaldehydes; Hemoglobin, Sickle; Humans; Point Mutation; Polymerization; Pyrazines; Pyrazoles; beta-Globins
PubMed: 32142671
DOI: 10.1016/j.cell.2020.01.019 -
Blood Apr 2021We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes...
We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.
Topics: Animals; Gene Knock-In Techniques; Hematopoietic Stem Cells; Humans; Interferon-alpha; Janus Kinase 2; Megakaryocytes; Mice; Mice, Transgenic; Myeloproliferative Disorders; Platelet Membrane Glycoprotein IIb; Point Mutation
PubMed: 33667305
DOI: 10.1182/blood.2020005563 -
Activity and Point Mutation G699V in PcoORP1 Confer Resistance to Oxathiapiprolin in Field Isolates.Journal of Agricultural and Food... Nov 2022The oxysterol-binding protein inhibitor oxathiapiprolin is a new fungicide for controlling oomycetes diseases. Besides, laboratory mutagenesis oxathiapiprolin-resistance...
The oxysterol-binding protein inhibitor oxathiapiprolin is a new fungicide for controlling oomycetes diseases. Besides, laboratory mutagenesis oxathiapiprolin-resistance among phytopathogenic oomycetes in the field remains unknown. Here, the sensitivity of 97 isolates to oxathiapiprolin was examined that were collected between 2011 and 2016. We obtained a baseline sensitivity with a mean EC value of 5.2639 × 10 μg mL. We showed that 6/32 isolates collected in Fujian Province from 2019 to 2020 were resistant to oxathiapiprolin without a significant fitness penalty on sporulation, vegetative growth, and virulence of the field isolates. The oxathiapiprolin resistance field isolates contained the point mutation glycine to valine at 699 in . The point mutation G699V was verified to confer resistance of to oxathiapiprolin using the CRISPR/Cas9 system. The mutation G699V decreased the binding affinity between oxathiapiprolin and . These results will improve our understanding of the mechanism of resistance to oxathiapiprolin under field conditions.
Topics: Phytophthora; Point Mutation; Plant Diseases; Hydrocarbons, Fluorinated; Fungicides, Industrial
PubMed: 36315898
DOI: 10.1021/acs.jafc.2c06707 -
BMC Evolutionary Biology Jul 2020Tumors are widely recognized to progress through clonal evolution by sequentially acquiring selectively advantageous genetic alterations that significantly contribute to...
BACKGROUND
Tumors are widely recognized to progress through clonal evolution by sequentially acquiring selectively advantageous genetic alterations that significantly contribute to tumorigenesis and thus are termned drivers. Some cancer drivers, such as TP53 point mutation or EGFR copy number gain, provide exceptional fitness gains, which, in time, can be sufficient to trigger the onset of cancer with little or no contribution from additional genetic alterations. These key alterations are called superdrivers.
RESULTS
In this study, we employ a Wright-Fisher model to study the interplay between drivers and superdrivers in tumor progression. We demonstrate that the resulting evolutionary dynamics follow global clonal expansions of superdrivers with periodic clonal expansions of drivers. We find that the waiting time to the accumulation of a set of superdrivers and drivers in the tumor cell population can be approximated by the sum of the individual waiting times.
CONCLUSIONS
Our results suggest that superdriver dynamics dominate over driver dynamics in tumorigenesis. Furthermore, our model allows studying the interplay between superdriver and driver mutations both empirically and theoretically.
Topics: Biological Evolution; Clonal Evolution; Disease Progression; Humans; Mutation; Neoplasms; Point Mutation; Time Factors
PubMed: 32689942
DOI: 10.1186/s12862-020-01647-y