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European Journal of Endocrinology Dec 2023
Topics: Humans; Cohort Studies; Retrospective Studies; Sequence Deletion; DNA, Mitochondrial
PubMed: 38039503
DOI: 10.1093/ejendo/lvad165 -
Journal of Infection and Chemotherapy :... Jun 2022Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance...
INTRODUCTION
Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance to the anti-fungal drug terbinafine (TRF) have also been isolated from human patients worldwide.
METHODS
In this study, we isolated a TRF-resistant strain (N99-2) of T. rubrum from a patient with recurrent tinea unguium. In vitro susceptibility of the clinical isolate to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LCZ) was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. To identify mutations, we compared the gene sequence of N99-2 to that of a TRF-susceptible strain of T. rubrum. Results; In N99-2, the minimum inhibitory concentrations were 32 mg/L for TRF, <0.03 mg/L for ITZ, <0.03 mg/L for RVZ, and <0.03 mg/L for LCZ. The squalene epoxidase (SQLE) gene sequence in N99-2 was determined to be 1467 bp in length, and it encoded a protein of 488 amino acids, beginning with a putative initiating methionine (ATG). The following mutations were identified from the SQLE of N99-2: L393F and Y394del.
CONCLUSIONS
This is the first report of the detection of a deletion mutation in SQLE in a TRF-resistant strain. The protein of SQLE is the target of TRF, and it is essential for cell membrane synthesis in dermatophytes. However, dermatophyte cells were found to undergo gene mutations to escape the effects of antifungal agents.
Topics: Amino Acid Sequence; Antifungal Agents; Drug Resistance, Fungal; Humans; Itraconazole; Microbial Sensitivity Tests; Onychomycosis; Sequence Deletion; Squalene Monooxygenase; Terbinafine; Trichophyton
PubMed: 35219578
DOI: 10.1016/j.jiac.2022.02.010 -
Virus Research Jan 2022Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in...
Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in nature. Phages are natural predators of bacteria controlling their population levels; however, their genomes carry several genes with unknown functions. In this work, Bacteriophage Recombineering of Electroporated DNA was used to assess the influence of deletion of a single gene with unknown function in the overall replication parameters of Salmonella phage PVP-SE2. Deletion of ORF_01, transcribed immediately after infection, reduced both the latent and rise periods by 5 min in PVP-SE2ΔORF_01 compared to the wild-type phage. A direct consequence of the deletion led to a smaller progeny release per infected cell by the mutant compared to the wild-type phage. Despite the difference in growth characteristics, the mutant phage remained infective towards exponentially growing cells. The mutation engineered endured for at least ten passages, showing that there is no reversion back to the wild-type sequence. This study provides proof of concept that methodologies used for phage engineering should always be complemented by phage growth characterization to assess whether a mutation can trigger undesirable characteristics.
Topics: Bacteriophages; Gene Deletion; Salmonella Phages; Salmonella enteritidis; Sequence Analysis, DNA
PubMed: 34902446
DOI: 10.1016/j.virusres.2021.198654 -
Molecular Reproduction and Development Jul 2021Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical...
Glands of the uterus are essential for the establishment of pregnancy in mice and their products regulate embryo implantation and stromal cell decidualization critical for pregnancy establishment. Forkhead box A2 (FOXA2) is expressed specifically in the glands and a critical regulator of their differentiation, development and function. Progesterone and FOXA2 regulate members of a serine proteinase gene family (Prss28 and Prss29). Here, CRISPR-Cas9 genome-editing was used to create mice with a heterozygous or homozygous deletion of Prss28 or/and Prss29 to determine their biological roles in uterine function. Female mice lacking Prss28 and Prss29 or both developed normally and were fertile without alterations in uterine histoarchitecture, uterine gland number, or and gene expression. Thus, Prss28 and Prss29 are dispensable for female fertility and do not impact endometrial gland development or uterine function mice.
Topics: Animals; CRISPR-Cas Systems; Endometrium; Female; Gene Editing; Gene Knockout Techniques; Male; Mice; Mice, Knockout; Pregnancy; Sequence Deletion; Serine Endopeptidases; Uterus
PubMed: 33973295
DOI: 10.1002/mrd.23473 -
Aging Clinical and Experimental Research Jul 2021Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species....
BACKGROUND
Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells.
AIMS
Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages.
METHODS
We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency.
RESULTS
Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach.
DISCUSSION/CONCLUSIONS
The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.
Topics: Adult; Aged; Aged, 80 and over; Aging; Animals; DNA, Mitochondrial; Humans; Male; Mice; Middle Aged; Mitochondria; Muscle Fibers, Skeletal; Muscle, Skeletal; Sequence Deletion; Young Adult
PubMed: 32965609
DOI: 10.1007/s40520-020-01698-7 -
Acta Neuropathologica Communications Jun 2022Desmoplastic infantile astrocytoma (DIA) is rare, cystic and solid tumor of infants usually found in superficial cerebral hemispheres. Although DIA is usually benign,...
Desmoplastic infantile astrocytoma (DIA) is rare, cystic and solid tumor of infants usually found in superficial cerebral hemispheres. Although DIA is usually benign, uncommon cases bearing malignant histological and aggressive clinical features have been described in the literature. We report a newborn patient who was diagnosed with a DIA and died postresection. Pathologic examination revealed that the main part of the tumor had benign features, but the internal region showed areas with a more aggressive appearance, with higher-proliferative cells, anaplastic GFAP positive cells with cellular polymorphism, necrosis foci, vascular hyperplasia with endothelial proliferation and microtrombosis. Genetic study, performed in both regions of the tumor, showed a BRAF V600E mutation and a homozygous deletion in PTEN, without changes in other relevant genes like EGFR, CDKN2A, TP53, NFKBIA, CDK4, MDM2 and PDGFRA. Although PTEN homozygous deletions are described in gliomas, the present case constitutes the first report of a PTEN mutation in a DIA, and this genetic feature may be related to the malignant behavior of a usually benign tumor. These genetic findings may point at the need of further and deeper genetic characterization of DIAs, in order to better understand the biology of this tumor and to obtain new prognostic approaches, a better clinical management and targeted therapies, especially in malignant cases of DIA.
Topics: Astrocytoma; Brain Neoplasms; Ganglioglioma; Homozygote; Humans; Mutation; PTEN Phosphohydrolase; Phenotype; Proto-Oncogene Proteins B-raf; Sequence Deletion
PubMed: 35725578
DOI: 10.1186/s40478-022-01392-x -
Journal of the Formosan Medical... Jan 2020Type 1 sialidosis is a rare autosomal recessive lysosomal storage disease caused by Neuraminidase 1 (NEU1) gene mutations. We report a type 1 sialidosis patient with a... (Review)
Review
BACKGROUND/PURPOSE
Type 1 sialidosis is a rare autosomal recessive lysosomal storage disease caused by Neuraminidase 1 (NEU1) gene mutations. We report a type 1 sialidosis patient with a novel deletion mutation in NEU1 and compared the phenotypes within different ethnicities.
METHODS
Targeted next generation sequencing and segregation analysis were performed to identify the causative gene mutation of the index patient. The clinical and electrophysiological characteristics of the patient were compared to those reported in previous studies of type 1 sialidosis from 1996 to 2019.
RESULTS
A 16-year-old boy presented with progressive onset of seizure, myoclonus, and ataxia since 5 years of age. Targeted next generation sequencing revealed the pathogenic missense variant c.544A>G (p.Ser182Gly) and the novel c.314_352del (p.A106_G118del) deletion in NEU1 in a compound heterozygote state. The leukocyte neuraminidase activity was significantly decreased (0.0323 nmol/mg protein/hour, normal reference: 0.326 ± 0.095 nmol/mg protein/hour). A total of 46 patients were identified in 18 reports from the literature. The most common symptoms were myoclonus (100%), followed by ataxia (88.3%) and seizure (72.5%). Notably, impaired cognition (50.0% vs. 21.7%, P = 0.04) and cherry-red spots (61.1% vs. 40.7%, P = 0.02) were less frequently reported in Asian patients than in Caucasian patients. Abnormal somatosensory evoked potentials with giant cortical waves and prolonged visual evoked potential latency were found consistently in Asian and Caucasian patients, and could be a surrogate marker of early diagnosis.
CONCLUSION
Our findings suggest a distinct phenotype of infrequent cherry-red spots and abnormal evoked potentials in Asian patients with type 1 sialidosis.
Topics: Adolescent; Humans; Male; Mucolipidoses; Mutation, Missense; Myoclonus; Neuraminidase; Phenotype; Seizures; Sequence Deletion
PubMed: 31371146
DOI: 10.1016/j.jfma.2019.07.017 -
Pathology Jun 2023Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for...
Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously.
Topics: Humans; Cyclin-Dependent Kinase Inhibitor p16; Oligodendroglioma; Homozygote; DNA Copy Number Variations; Sequence Deletion; Gene Deletion; Glioma; Biomarkers; Phosphorylases; Astrocytoma; Brain Neoplasms; Isocitrate Dehydrogenase; Mutation
PubMed: 37032198
DOI: 10.1016/j.pathol.2023.01.005 -
PloS One 2024Quorum sensing can induce density-dependent gene expressions that cause various problems. For quorum-sensing inhibition, fundamental solutions such as gene manipulation...
Quorum sensing can induce density-dependent gene expressions that cause various problems. For quorum-sensing inhibition, fundamental solutions such as gene manipulation are required, and acyl-homoserine lactone synthase (AHL synthase), which synthesizes the universal quorum-sensing signal of gram-negative bacteria, can be used as a target. In this study, researchers synthesized His-tagged AHL synthase and its deletion mutant that lacks the active site and compared their biochemical characteristics. His-YpeI, the 6x His-tagged AHL synthase of Serratia fonticola, and His-ΔYpeI, its deletion mutant, were designed, and their property conservation were examined using in silico projection tools. For in vitro synthesis of enzymes, the His-YpeI CFPS template was synthesized by in vitro gene synthesis, and the His-ΔYpeI CFPS template was obtained by deletion PCR. CFPS was performed and the products were purified with the 6x His-tag. The enzymes' properties were compared using an enzymatic assay. The bioinformatic analysis confirmed the conservation of biochemical properties between 6x His-tagged and untagged enzymes, including helix-turn-helix interactions, hydropathy profiles, and tertiary structure between His-YpeI and YpeI and between His-ΔYpeI and ΔYpeI. His-YpeI and His-ΔYpeI synthesized by CFPS were found to have the expected molecular weights and demonstrated distinct differences in enzyme activity. The analyzed enzymatic constants supported a significant decrease in substrate affinity and reaction rate as a result of YpeI's enzyme active site deletion. This result showed that CFPS could be used for in vitro protein synthesis, and quorum sensing could be inhibited at the enzymatic level due to the enzyme active site's deletion mutation.
Topics: Quorum Sensing; Acyltransferases; Sequence Deletion; Serratia; Bacterial Proteins; Catalytic Domain; Amino Acid Sequence; Ligases
PubMed: 38820426
DOI: 10.1371/journal.pone.0304331 -
Thrombosis and Haemostasis Jul 2022Tissue factor pathway inhibitor (TFPI) is an anticoagulant that inhibits factor VIIa and Xa in the blood coagulation pathways. TFPI contains three Kunitz domains, K1,...
Tissue factor pathway inhibitor (TFPI) is an anticoagulant that inhibits factor VIIa and Xa in the blood coagulation pathways. TFPI contains three Kunitz domains, K1, K2, and K3. K1 and K2 inhibit factor VIIa and Xa, respectively. However, the regulation of TFPI is poorly studied. Since zebrafish has become an alternate model to discover novel actors in hemostasis, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of in zebrafish using reverse transcription polymerase chain reaction. We then performed piggyback knockdowns of and found increased coagulation activity in knockdown. We then created a deletion mutation in locus using the CRISPR/Cas9 method. The homozygous deletion mutants showed increased coagulation activities similar to that found in knockdown. Taken together, our data suggest that is a negative regulator for zebrafish coagulation, and silencing it leads to thrombotic phenotype. Also, the zebrafish knockout model could be used for reversing this thrombotic phenotype to identify antithrombotic novel factors by the genome-wide piggyback knockdown method.
Topics: Animals; Factor VIIa; Factor Xa; Homozygote; Lipoproteins; Sequence Deletion; Zebrafish
PubMed: 34918310
DOI: 10.1055/a-1723-4075