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Mental Retardation and Developmental... 2007Past autism research has often been dedicated to tracing the causes of the disorder to a localized neurological abnormality, a single functional network, or a single... (Review)
Review
Past autism research has often been dedicated to tracing the causes of the disorder to a localized neurological abnormality, a single functional network, or a single cognitive-behavioral domain. In this review, I argue that autism is a "distributed disorder" on various levels of study (genetic, neuroanatomical, neurofunctional, behavioral). "Localizing" models are therefore not promising. The large array of potential genetic risk factors suggests that multiple (or all) emerging functional brain networks are affected during early development. This is supported by widespread growth abnormalities throughout the brain. Interactions during development between affected functional networks and atypical experiential effects (associated with atypical behavior) in children with autism further complicate the neurological bases of the disorder, resulting in an "exponentially distributed" profile. Promising approaches to a better characterization of neural endophenotypes in autism are provided by techniques investigating white matter and connectivity, such as MR spectroscopy, diffusion-tensor imaging (DTI), and functional connectivity MRI. According to a recent hypothesis, the autistic brain is generally characterized by "underconnectivity." However, not all findings are consistent with this view. The concepts and methodology of functional connectivity need to be refined and results need to be corroborated by anatomical studies (such as DTI tractography) before definitive conclusions can be drawn.
Topics: Adolescent; Adult; Autistic Disorder; Brain; Fragile X Mental Retardation Protein; Humans; Magnetic Resonance Imaging; Nerve Net; Neuronal Plasticity; Point Mutation
PubMed: 17326118
DOI: 10.1002/mrdd.20141 -
BMC Molecular and Cell Biology Jun 2022Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that promotes cell proliferation and immunomodulation in untransformed...
BACKGROUND
Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that promotes cell proliferation and immunomodulation in untransformed cells and maintains stemness of transformed cells, facilitating invasion and metastasis. Numerous point mutations in the STAT3 protein have been identified that drive malignancy in various tumor entities. The missense mutation D427H localized in the STAT3 DNA-binding domain has been previously reported in patients with NK/T cell lymphomas. To assess the biological activity of this missense mutation, we compared the STAT3-D427H mutant to wild-type (WT) protein as well as the known hyper-active mutant F174A.
RESULTS
Although previously reported as an activating mutation, the STAT3-D427H mutant neither showed elevated cytokine-induced tyrosine phosphorylation nor altered nuclear accumulation, as compared to the WT protein. However, the D427H mutant displayed enhanced binding to STAT-specific DNA-binding sites but a reduced sequence specificity and dissociation rate from DNA, which was demonstrated by electrophoretic mobility shift assays. This observation is consistent with the phenotype of the homologous E421K mutation in the STAT1 protein, which also displayed enhanced binding to DNA but lacked a corresponding increase in transcriptional activity.
CONCLUSIONS
Based on our data, it is unlikely that the D427H missense mutation in the STAT3 protein possesses an oncogenic potential beyond the WT molecule.
Topics: DNA; Humans; Lymphoma; Point Mutation; STAT3 Transcription Factor; Signal Transduction
PubMed: 35752777
DOI: 10.1186/s12860-022-00422-9 -
ACS Synthetic Biology Oct 2017Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of...
Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.
Topics: Bacteriophage T4; CRISPR-Cas Systems; Genetic Engineering; Plasmids; Point Mutation
PubMed: 28657724
DOI: 10.1021/acssynbio.7b00179 -
Genetics and Molecular Research : GMR Mar 2016The aim of this study was to identify changes in the base sequence of the upstream regulatory region of the transthyretin (TTR) gene. Whole-blood DNA was extracted from...
The aim of this study was to identify changes in the base sequence of the upstream regulatory region of the transthyretin (TTR) gene. Whole-blood DNA was extracted from ten subjects belonging to a family with familial amyloidosis vitreoretinopathy; the upstream regulatory sequence was amplified by polymerase chain reaction, detected by gel electrophoresis, and sequenced. The DNA sequence of the upstream regulatory region of the TTR gene was successfully sequenced, and a point mutation (-743A→T) was identified in six of the ten blood samples: four patients and two family members without disease incidence. Therefore, a point mutation was identified in the upstream regulatory region of the TTR gene in a Han Chinese family with familial vitreous amyloidosis.
Topics: Adolescent; Adult; Amyloidosis, Familial; Exons; Female; Humans; Male; Middle Aged; Pedigree; Point Mutation; Prealbumin; Young Adult
PubMed: 27051017
DOI: 10.4238/gmr.15016926 -
MBio Nov 2017The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties...
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.
Topics: Directed Molecular Evolution; Genetic Code; Models, Genetic; Point Mutation; Synthetic Biology
PubMed: 29138304
DOI: 10.1128/mBio.01654-17 -
Scientific Reports Jun 2023Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and...
Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and activating more than a dozen coagulation factors and resulting in the formation of a fibrin clot that ceases the bleeding. In this process, the Coagulation factor V (FV) is a master regulator, coordinating critical steps of this process. Mutations to this factor result in spontaneous bleeding episodes and prolonged hemorrhage after trauma or surgery. Although the role of FV is well characterized, it is unclear how single-point mutations affect its structure. In this study, to understand the effect of mutations, we created a detailed network map of this protein, where each node is a residue, and two residues are connected if they are in close proximity in the three-dimensional structure. Overall, we analyzed 63 point-mutations from patients and identified common patterns underlying FV deficient phenotypes. We used structural and evolutionary patterns as input to machine learning algorithms to anticipate the effects of mutations and anticipated FV-deficiency with fair accuracy. Together, our results demonstrate how clinical features, genetic data and in silico analysis are converging to enhance treatment and diagnosis of coagulation disorders.
Topics: Humans; Factor V; Mutation; Point Mutation; Algorithms; Biological Evolution
PubMed: 37308572
DOI: 10.1038/s41598-023-36528-z -
G3 (Bethesda, Md.) May 2018The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has...
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the or marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.
Topics: Base Sequence; CRISPR-Cas Systems; Cloning, Molecular; DNA Repair; Gene Editing; Gene Knock-In Techniques; Point Mutation; Schizosaccharomyces; Sequence Deletion; Temperature
PubMed: 29703785
DOI: 10.1534/g3.118.200164 -
Journal For Immunotherapy of Cancer Oct 2020Tumor relapse due to mutation in can hinder the efficacy of chimeric antigen receptor (CAR)-T cell therapy. Herein, we focused on lymphoma patients whose B cells...
BACKGROUND
Tumor relapse due to mutation in can hinder the efficacy of chimeric antigen receptor (CAR)-T cell therapy. Herein, we focused on lymphoma patients whose B cells exhibited a point mutation in of B cells after CAR-T cell infusion.
METHODS
The CAR-T and CD19 B cells from peripheral blood or bone marrow were assessed using flow cytometry. Genome sequencing was conducted to identify the molecular characteristics of CAR-T and CD19 B cells from pre-relapse and postrelapse samples. CD19 in CARs comprising single chain fragments variable (scFV) antibody with FMC63 or 21D4 was constructed. The cytotoxic efficacy of CAR-T cells was also evaluated via in vitro and in vivo experiments.
RESULTS
A patient with high-grade B cell lymphoma exhibited complete response, but the lymphoma relapsed in her left breast at 6 months after CAR (FMC63)-T cell infusion. A mutation was found in exon 3 of (p.163. R-L) in malignant B cells of the patient. In two lymphoma patients who exhibited resistance to CAR-T cell therapy, a mutation was detected in exon 3 of (p.174. L-V). Functional analysis revealed that FMC63 CAR-T cells exhibited antitumor ability against wild-type CD19 cells but were unable to eradicate these two types of mutated CD19 cells. Interestingly, 21D4 CAR-T cells were potentially capable of eradicating these mutated CD19 cells and exhibiting high antitumor capacity against CD19 cells with loss of exon 1, 2, or 3.
CONCLUSIONS
These findings suggest that point mutation can facilitate immune escape from CAR-T cell therapy and that alternative CAR-T cells can effectively eradicate the mutated B cells, providing an individualized therapeutic approach for lymphoma patients showing relapse.
Topics: Animals; Antigens, CD19; Cell- and Tissue-Based Therapy; Female; Humans; Immunotherapy, Adoptive; Lymphoma, B-Cell; Male; Mice; Mice, SCID; Middle Aged; Point Mutation; Young Adult
PubMed: 33023981
DOI: 10.1136/jitc-2020-001150 -
PLoS Computational Biology Jun 2017Natural protein sequences contain a record of their history. A common constraint in a given protein family is the ability to fold to specific structures, and it has been...
Natural protein sequences contain a record of their history. A common constraint in a given protein family is the ability to fold to specific structures, and it has been shown possible to infer the main native ensemble by analyzing covariations in extant sequences. Still, many natural proteins that fold into the same structural topology show different stabilization energies, and these are often related to their physiological behavior. We propose a description for the energetic variation given by sequence modifications in repeat proteins, systems for which the overall problem is simplified by their inherent symmetry. We explicitly account for single amino acid and pair-wise interactions and treat higher order correlations with a single term. We show that the resulting evolutionary field can be interpreted with structural detail. We trace the variations in the energetic scores of natural proteins and relate them to their experimental characterization. The resulting energetic evolutionary field allows the prediction of the folding free energy change for several mutants, and can be used to generate synthetic sequences that are statistically indistinguishable from the natural counterparts.
Topics: Energy Transfer; Evolution, Chemical; Models, Chemical; Models, Molecular; Point Mutation; Protein Conformation; Protein Folding; Proteins; Repetitive Sequences, Amino Acid; Sequence Analysis, Protein; Structure-Activity Relationship
PubMed: 28617812
DOI: 10.1371/journal.pcbi.1005584 -
Nature Communications May 2020Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically...
Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.
Topics: Animals; Caenorhabditis elegans; DNA Damage; DNA Repair; Genomics; Humans; Mutation; Point Mutation
PubMed: 32358516
DOI: 10.1038/s41467-020-15912-7