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Current Opinion in Microbiology Dec 2019The fungus Candida albicans exists as a prevalent commensal and an important opportunistic pathogen that can infect multiple niches of its human host. Recent studies... (Review)
Review
The fungus Candida albicans exists as a prevalent commensal and an important opportunistic pathogen that can infect multiple niches of its human host. Recent studies have examined the diploid genome of C. albicans by performing both short-term microevolution studies and comparative genomics on collections of clinical isolates. Common mechanisms driving genome dynamics include accumulation of point mutations, loss of heterozygosity (LOH) events, large-scale chromosomal rearrangements, and even ploidy change, with important consequences for both drug resistance and host adaptation. Evidence for recombination between C. albicans lineages also highlights a role for (para)sex in shaping the species population structure. Ongoing work will continue to define the contributions of genome evolution to phenotypic variation and the role of host pressures in driving adaptive processes.
Topics: Adaptation, Biological; Candida albicans; Candidiasis; Drug Resistance, Fungal; Evolution, Molecular; Gene Rearrangement; Genome; Humans; Ploidies; Point Mutation; Virulence
PubMed: 31176092
DOI: 10.1016/j.mib.2019.05.001 -
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 -
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 -
BMC Bioinformatics Oct 2022RNA deleterious point mutation prediction was previously addressed with programs such as RNAmute and MultiRNAmute. The purpose of these programs is to predict a global...
BACKGROUND
RNA deleterious point mutation prediction was previously addressed with programs such as RNAmute and MultiRNAmute. The purpose of these programs is to predict a global conformational rearrangement of the secondary structure of a functional RNA molecule, thereby disrupting its function. RNAmute was designed to deal with only single point mutations in a brute force manner, while in MultiRNAmute an efficient approach to deal with multiple point mutations was developed. The approach used in MultiRNAmute is based on the stabilization of the suboptimal RNA folding prediction solutions and/or destabilization of the optimal folding prediction solution of the wild type RNA molecule. The MultiRNAmute algorithm is significantly more efficient than the brute force approach in RNAmute, but in the case of long sequences and large m-point mutation sets the MultiRNAmute becomes exponential in examining all possible stabilizing and destabilizing mutations.
RESULTS
An inherent limitation in the RNAmute and MultiRNAmute programs is their ability to predict only substitution mutations, as these programs were not designed to work with deletion or insertion mutations. To address this limitation we herein develop a very fast algorithm, based on suboptimal folding solutions, to predict a predefined number of multiple point deleterious mutations as specified by the user. Depending on the user's choice, each such set of mutations may contain combinations of deletions, insertions and substitution mutations. Additionally, we prove the hardness of predicting the most deleterious set of point mutations in structural RNAs.
CONCLUSIONS
We developed a method that extends our previous MultiRNAmute method to predict insertion and deletion mutations in addition to substitutions. The additional advantage of the new method is its efficiency to find a predefined number of deleterious mutations. Our new method may be exploited by biologists and virologists prior to site-directed mutagenesis experiments, which involve indel mutations along with substitutions. For example, our method may help to investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary structure.
Topics: INDEL Mutation; Mutation; Point Mutation; RNA; Sequence Analysis, RNA
PubMed: 36241988
DOI: 10.1186/s12859-022-04943-0 -
Methods in Cell Biology 2020The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for...
The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations in mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: single-large-scale rearrangements and point mutations.
Topics: DNA Mutational Analysis; DNA, Mitochondrial; Gene Rearrangement; Genome, Mitochondrial; High-Throughput Nucleotide Sequencing; Humans; Mutation; Point Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 32183969
DOI: 10.1016/bs.mcb.2019.11.009 -
Stem Cell Research May 2021When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point...
When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-Puro cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-Puro cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.
Topics: CRISPR-Cas Systems; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Homologous Recombination; Humans; Induced Pluripotent Stem Cells; Mutation; Point Mutation
PubMed: 33857832
DOI: 10.1016/j.scr.2021.102332 -
Scientific Reports Feb 2023An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6...
An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide. It has been shown that a point mutation of glutamate 69 to alanine (E69A) allows OlyA6 to bind to cholesterol-free sphingomyelin. Using artificial lipid membranes and mammalian MDCK cells, we show that this mutation significantly enhances the interaction of OlyA6 with sphingomyelin and CPE, and allows recognition of these sphingolipids even in the absence of cholesterol. Our results suggest that OlyA6 mutant E69A could serve as complementary tool to detect and study cholesterol-associated and free sphingomyelin or CPE in membranes. However, the mutation does not improve the membrane-permeabilizing activity after addition of pleurotolysin B, which was confirmed in toxicity tests on insect and mammalian cell lines, and on Colorado potato beetle larvae.
Topics: Animals; Sphingomyelins; Point Mutation; Cell Membrane; Cholesterol; Insecta; Membranes, Artificial; Mammals
PubMed: 36750638
DOI: 10.1038/s41598-023-28949-7 -
BMC Microbiology Feb 2022Amoxicillin-resistant Helicobacter pylori (H. pylori) strains seem to have increased over time in Vietnam. This threatens the effectiveness of H. pylori eradication...
BACKGROUND
Amoxicillin-resistant Helicobacter pylori (H. pylori) strains seem to have increased over time in Vietnam. This threatens the effectiveness of H. pylori eradication therapies with this antibiotic. This study aimed to investigate the prevalence of primary resistance of H. pylori to amoxicillin and to assess its association with pbp1A point mutations in Vietnamese patients.
MATERIALS AND METHODS
Naive patients who presented with dyspepsia undergoing upper gastrointestinal endoscopy were recruited. Rapid urease tests and PCR assays were used to diagnose H. pylori infection. Amoxicillin susceptibility was examined by E-tests. Molecular detection of the mutant pbp1A gene conferring amoxicillin resistance was carried out by real-time PCR followed by direct sequencing of the PCR products. Phylogenetic analyses were performed using the Tamura-Nei genetic distance model and the neighbor-joining tree building method.
RESULTS
There were 308 patients (46.1% men and 53.9% women, p = 0.190) with H. pylori infection. The mean age of the patients was 40.5 ± 11.4 years, ranging from 18 to 74 years old. The E-test was used to determine the susceptibility to amoxicillin (minimum inhibitory concentration (MIC) ≤ 0.125 μg/ml) in 101 isolates, among which the rate of primarily resistant strains to amoxicillin was 25.7%. Then, 270 sequences of pbp1A gene fragments were analysed. There were 77 amino acid substitution positions investigated, spanning amino acids 310-596, with the proportion varying from 0.4 to 100%. Seven amino acid changes were significantly different between amoxicillin-sensitive (Amox) and amoxicillin-resistant (Amox) samples, including Phe to Leu (p < 0.001), Ser to Arg (p < 0.001), Glu/Asn (p = 0.009), Val to Met (p = 0.021), Phe to Val (p < 0.001), Asp to Glu (p = 0.044), and Ser/Ala/Gly (p = 0.001). Phylogenetic analyses suggested that other molecular mechanisms might contribute to amoxicillin resistance in H. pylori in addition to the alterations in PBP1A.
CONCLUSIONS
We reported the emergence of amoxicillin-resistant Helicobacter pylori strains in Vietnam and new mutations statistically associated with this antimicrobial resistance. Additional studies are necessary to identify the mechanisms contributing to this resistance in Vietnam.
Topics: Adolescent; Adult; Aged; Amino Acid Substitution; Amoxicillin; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance; Female; Gastrointestinal Tract; Helicobacter Infections; Helicobacter pylori; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Penicillin-Binding Proteins; Point Mutation; Vietnam; Young Adult
PubMed: 35114945
DOI: 10.1186/s12866-022-02463-8