mutation assay
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PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261900
Author(s):  
Margitta Dziwenka ◽  
Laurie Dolan ◽  
Jason Mitchell

VOHO Hemp Oil (Verdant Nature LLC (in collaboration with HempFusion)) is an extract of the aerial parts of hemp (Cannabis sativa L) manufactured using a supercritical CO2 extraction process. The results of four safety studies are reported here including a bacterial reverse mutation assay, an in vivo mammalian micronucleus study, a maximum tolerated dose study in rats and a 90-day repeat dose subchronic toxicity study in rats. VOHO Hemp oil can contain up to 30% phytocannabinoids and less than 0.2% is tetrahydrocannabinol (THC). VOHO Hemp Oil was found to be non-mutagenic in the bacterial reverse mutation assay and was negative for inducing micronuclei in the rat bone marrow micronucleus assay. The maximum tolerated dose in male and female Wistar rats was 2250 mg/kg bw/day. A 90-day repeat dose study was conducted in male and female Wistar rats according to OECD Guideline 408 and included a 21-day recovery period. The doses used in the study were 0, 25, 90 and 324 mg/kg bw per day in the main study, and in the recovery phase a control and 324 mg/kg bw/day group were included. One mortality was reported during the study, a high dose female, and test substance-related adverse clinical signs were reported in the high dose group. Other test substance-related changes noted in the high dose group included changes in body weights, activated partial thromboplastin time (APTT) values, and in absolute and relative organ weights. Based on the results of the study, the no observed adverse effect level (NOAEL) for VOHO Hemp Oil was determined to be 90 mg/kg bw/day in both male and female Wistar rats.


2021 ◽  
Author(s):  
Mei-Qin Zhuo ◽  
Jun Chen ◽  
Mei-Li Wu ◽  
Wen-biao Wang

Abstract In this study, the transcriptional regulation of PI3KC3 by three transcript factors (PPARγ, PPARα and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5’-deletion assay, overexpression assay, site-mutation assay and electrophoretic mobility shift assay suggested that PPARα, PPARγ and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG), NEFA content, the mRNA level of pparα, pparγ, stat3 and dnmt3b, the protein level of PPARα, PPARγ and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.


2021 ◽  
Author(s):  
Cuong Thanh Le ◽  
Erin P Prince ◽  
Derek S Sarovich ◽  
Thu Thi Anh Nguyen ◽  
Hung VuKhac ◽  
...  

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.


2021 ◽  
Author(s):  
Danielle E. Madden ◽  
Kate L. McCarthy ◽  
Scott C. Bell ◽  
Olusola Olagoke ◽  
Timothy Baird ◽  
...  

Antimicrobial resistance (AMR) is an ever-increasing global health concern. Here, we developed two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I, D87N, D87Y, and D87H, the predominant causes of fluoroquinolone AMR in Pseudomonas aeruginosa. SYBR-MAMAs were tested on 85 isolates, 45 with intermediate/full ciprofloxacin resistance. Assays identified the correct phenotype in 84% intermediate/full resistant and 100% sensitive strains. Clinical implementation of our rapid SYBR-MAMAs will permit timely treatment alterations to improve patient outcomes.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4622-4622
Author(s):  
Durga Prasad Dash ◽  
David Dinauer ◽  
Michael Janasik

Abstract Recently in June 2021, the Food and Drug Administration approved avapritinib (Ayvakit™) for adult patients with advanced systemic mastocytosis (AdvSM), including patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL). Mastocytosis is a heterogeneous, neoplastic disorder characterized by infiltration of abnormal mast cells in one or more organs. Mastocytosis subtypes are defined by disease distribution and the clinical features include, cutaneous mastocytosis (CM), where mast cell infiltration is confined to the skin, and systemic mastocytosis (SM) in which at least one extracutaneous organ is involved, with or without evidence of skin lesions. CM is most common in pediatric patients whereas SM typically presents in adulthood. The presence of somatic, activating mutations in KIT codon 816, predominantly D816V (A2447T), can be identified in the mast cells of 95% or more of patients with systemic mastocytosis (SM) and represent clonal markers in the disease. KIT is located on chromosome 4q12 and encodes for the mast/stem cell growth factor receptor, a type III receptor tyrosine kinase. Detection of KIT D816 mutations serve as a World Health Organization (WHO) minor criterion for the diagnosis of systematic mastocytosis and appear to confer relative resistance to tyrosine kinase inhibitors. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has developed a highly sensitive clinical test for the detection of KIT D816 mutations and molecular diagnosis of systemic mastocytosis patients. The KIT D816 mutation assay is a unique laboratory developed test based on highly sensitive allele specific PCR and its performance characteristics were determined by our laboratory. The sensitivity of the KIT D816 assay is 0.25% allele proportion. The specificity for the D816V mutation is > 99%. Systemic Mastocytosis (SM) is classified as myeloid neoplasm by WHO, however SM may be missed in suspected myeloid neoplasm work up by conventional sequencing technology including NGS which might not be able to achieve higher sensitivity. It is important to have a highly sensitive KIT D816 mutation analysis assay to detect low levels of KIT D816 allele burden that may be present in blood or bone marrow especially at early stage disease. Our highly sensitive laboratory developed KIT D816 assay based on allele specific PCR with a limit of detection of 0.25% could help physicians identify SM patients and clinically manage the disease. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Recently FDA approved Avapritinib for adult patients with advanced systemic mastocytosis (AdvSM), including patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL).


2021 ◽  
Author(s):  
Maud Hamadou ◽  
Jonathan Lopez ◽  
Nazim Benzerdjeb ◽  
Christine Cugnet‐Anceau ◽  
Gwenaelle Schnoering ◽  
...  

2021 ◽  
Vol 03 (02) ◽  
pp. e77-e85
Author(s):  
Chang-Hui Zhou ◽  
Chun-Rong Yu ◽  
Peng-Cheng Huang ◽  
Ruo-Wan Li ◽  
Jing-Ting Wang ◽  
...  

AbstractThe X-linked PIG-A gene is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD59 and CD55). In recent years, in vitro PIG-A assay has been established based on the high conservation of PIG-A/Pig-a loci among different species and the large data from the in vivo system. The purpose of this study was to extend the approach for PIG-A mutation assessment to in vitro human B-lymphoblastoid TK6 cells by detecting the loss of GPI-linked CD55 and CD59 proteins. TK6 cells were treated with three mutagens 7,12-dimethylbenz[a]anthracene (DMBA), N-ethyl-N-nitrosourea (ENU), etoposide (ETO), and two nonmutagens: cadmium chloride (CdCl2) and sodium chloride (NaCl). The mutation rate of PIG-A gene within TK6 cells was determined on the 11th day with flow cytometry analysis for the negative frequencies of CD55 and CD59. The antibodies used in this production were APC mouse-anti-human CD19 antibody, PE mouse anti-human CD55 antibody, PE mouse anti-human CD59 antibody, and nucleic acid dye 7-AAD. An immunolabeling method was used to reduce the high spontaneous level of preexisting PIG-A mutant cells. Our data suggested that DMBA-, ENU-, and ETO-induced mutation frequency of PIG-A gene was increased by twofold compared with the negative control, and the effects were dose-dependent. However, CdCl2 and NaCl did not significantly increase the mutation frequency of PIG-A gene, with a high cytotoxicity at a dose of 10 mmol/L. Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pauline Gilson ◽  
Chloé Saurel ◽  
Julia Salleron ◽  
Marie Husson ◽  
Jessica Demange ◽  
...  

AbstractThe assessment of EGFR mutations is recommended for the management of patients with non-small cell lung cancer (NSCLC). Presence of EGFR mutation is associated with response or resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI). Liquid biopsy is nowadays widely used for the detection of resistance to EGFR-TKI. We evaluated here the performance of the Idylla ctEGFR mutation assay for the detection of EGFR mutations in circulating tumour DNA (ctDNA) in plasma from patients with NSCLC. Previously characterized plasma samples from 38 patients with NSCLC were analysed using 2 different analytical conditions (C1 and C2). The limit of detection (LOD) was evaluated using 2 mL of healthy donor plasma spiked with commercial DNA controls. Overall agreement, sensitivity and specificity were 92.1%, 86.7% and 95.7% for C1 condition respectively and 94.7%, 86.7% and 100% for C2 condition respectively. The T790M secondary resistance mutation was detected in two samples out of 3. The Idylla system was able to detect the exon 19 deletion from 6 copies/mL and up to 91 copies/mL for the G719S mutation. These results support that the Idylla ctEGFR mutation assay is a rapid option for the detection of EGFR hotspots mutations in plasma samples, however a particular attention is needed for its interpretation.


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