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STAR Protocols Sep 2023Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been...
Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023)..
Topics: Adult; Animals; Mice; Infant, Newborn; Humans; Microglia; Brain; Disease Models, Animal; Pluripotent Stem Cells; Point Mutation
PubMed: 37516973
DOI: 10.1016/j.xpro.2023.102490 -
The Journal of Biological Chemistry Feb 2022The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR mice with reduced dimerization propensity due...
The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR) have previously helped to define the functions of GR monomers and dimers. Since GR retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.
Topics: Animals; Dexamethasone; Glucocorticoids; Ligands; Mice; Point Mutation; Receptors, Glucocorticoid
PubMed: 35007536
DOI: 10.1016/j.jbc.2022.101574 -
Genes Oct 2021The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb...
The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb anomalies, and seizure disorders. OTUD6B- and ZMIZ1-related ID are inherited in autosomal recessive and autosomal dominant patterns, respectively. We report a 5-year-old girl with developmental delay, facial phenotypes resembling Williams syndrome, and cardiac defects. The patient also had terminal broadening of the fingers and polydactyly. Cytogenomic microarray (CMA), whole exome sequencing (WES), and mRNA analysis were performed. The CMA showed a paternally inherited 0.118 Mb deletion of 8q21.3, chr8:92084087-92202189, with OTUD6B involved. The WES identified a hemizygous OTUD6B variant, c.873delA (p.Lys291AsnfsTer3). The mother was heterozygous for this allele. The WES also demonstrated a heterozygous ZMIZ1 variant, c.1491 + 2T > C, in the patient and her father. This ZMIZ1 variant yielded exon 14 skipping, as evidenced by mRNA study. We suggest that Williams syndrome-like phenotypes, namely, periorbital edema, hanging cheek, and long and smooth philtrum represent expanded phenotypes of OTUD6B-related ID. Our data expand the genotypic spectrum of OTUD6B- and ZMIZ1-related disorders. This is the first reported case of a compound heterozygote featuring point mutation, chromosomal microdeletion of OTUD6B, and the unique event of OTUD6B, coupled with ZMIZ1 variants.
Topics: Alleles; Child, Preschool; Chromosome Deletion; Endopeptidases; Exome; Female; Genetic Predisposition to Disease; Heterozygote; Humans; Infant; Intellectual Disability; Male; Pedigree; Phenotype; Point Mutation; Transcription Factors; Exome Sequencing
PubMed: 34680978
DOI: 10.3390/genes12101583 -
Journal of Molecular Modeling Mar 2023Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
INTRODUCTION
Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
METHOD
The present study is focused on mutation profiling of CCHFV L segment and phylogenetic clustering of protein dataset into six CCHFV genotypes.
RESULTS
Phylogenetic tree rooted with NCBI reference sequence (YP_325663.1) indicated less divergence from genotype III and the sequences belonging to same genotypes have shown less divergence among each other. Mutation frequency at 729 mutated positions was calculated and 563, 49, 33, 46 and 38 amino acid positions were found to be mutated at mutation frequency intervals of 0-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1.0 respectively. Thirty-eight highly frequent mutations (0.81-1.0 interval) were found in all genotypes and mapping in L segment (encoded for RdRp) revealed four mutations (V2074I, I2134T/A, V2148A and Q2695H/R) in catalytic site domain and no mutation in OTU domain. Molecular dynamic simulation and in silico analysis showed that catalytic site domain displayed large deviation and fluctuation upon introduction of these point mutations.
CONCLUSION
Overall study provides strong evidence that OTU domain is highly conserved and less prone to mutation whereas point mutations recorded in catalytic domain have affected the stability of protein and were found to be persistent in the large population.
Topics: Catalytic Domain; Phylogeny; Mutation; Point Mutation; RNA
PubMed: 36877258
DOI: 10.1007/s00894-023-05487-7 -
Molecules (Basel, Switzerland) Aug 2021Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been... (Review)
Review
Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.
Topics: Biosensing Techniques; Electrochemistry; Electrons; Oxidation-Reduction; Point Mutation; Protein Folding; Proteins
PubMed: 34443538
DOI: 10.3390/molecules26164950 -
Methods in Molecular Biology (Clifton,... 2021TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They...
TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process.
Topics: DNA End-Joining Repair; Gene Editing; Genome, Fungal; Point Mutation; Software; Transcription Activator-Like Effector Nucleases; Yarrowia
PubMed: 33847980
DOI: 10.1007/978-1-0716-1414-3_2 -
The Tohoku Journal of Experimental... Aug 2022Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human...
Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.
Topics: Adenoviruses, Human; Genome, Viral; INDEL Mutation; Point Mutation; SARS-CoV-2
PubMed: 35705320
DOI: 10.1620/tjem.2022.J049 -
Frontiers in Immunology 2022Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer...
Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6 cell was enriched in skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in deficient patients. Therefore, our results demonstrate that point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.
Topics: Animals; Homeostasis; Hyperpigmentation; Imiquimod; Mice; Mice, Knockout; Point Mutation
PubMed: 35769463
DOI: 10.3389/fimmu.2022.728455 -
HLA Oct 2022The novel HLA-DQB1*04:02:01:16Q allele showing a point mutation in the intron 3. (Review)
Review
The novel HLA-DQB1*04:02:01:16Q allele showing a point mutation in the intron 3.
Topics: Alleles; HLA-DQ beta-Chains; Humans; Introns; Point Mutation
PubMed: 35642675
DOI: 10.1111/tan.14697 -
Journal of the American Chemical Society Feb 2020Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes...
Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
Topics: Catalytic Domain; Glycoside Hydrolases; Phosphorylases; Point Mutation
PubMed: 31917561
DOI: 10.1021/jacs.9b09655