Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative ...
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Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 mu L of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.
The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms ...
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The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 x 10(6) copy/mg total RNA), AKR1C3 (1.3 x 10(6)), AKR1C1 (1.3 x 10(6)), CBR1 (9.7 x 10(5)), and HSD11B1 (1.1 x 10(6)) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver. (C) 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Enzymes of the aldo-keto reductase (AKR) and short-chain dehydro-genase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be invol...
Enzymes of the aldo-keto reductase (AKR) and short-chain dehydro-genase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be involved in the reduction of a compound, and the enzyme(s) that are responsible for the reaction in the hu-man liver have not been elucidated. The purpose of this study was to quantitatively evaluate the contribution of each isoform to reduction reactions in the human liver. Recombinant cytosolic isoforms were constructed, i.e., AKR1C1, AKR1C2, AKR1C3, AKR1C4, and carbonyl reductase 1 (CBR1), and a microsomal isoform, 11/3-hydroxysteroid dehydrogenase type 1 (HSD11B1), and their contributions to the reduction of 10 compounds were examined by extrapolating the relative expression of each reduc-tase protein in human liver preparations to recombinant systems quantified by liquid chromatography-;mass spectrometry. The re-ductase activities for acetohexamide, doxorubicin, haloperidol, loxoprofen, naloxone, oxcarbazepine, and pentoxifylline were predominantly catalyzed by cytosolic isoforms, and the sum of the contributions of individual cytosolic reductases was almost 100%. Interestingly, AKR1C3 showed the highest contribution to acetohexamide and loxoprofen reduction, although previous studies have revealed that CBR1 mainly metabolizes them. The reductase activities of bupropion, ketoprofen, and tolperisone were catalyzed by microsomal isoform(s), and the contributions of HSD11B1 were calculated to be 41%, 32%, and 104%, respec-tively. To our knowledge, this is the first study to quantitatively evaluate the contribution of each reductase to the reduction of drugs in the human liver. SIGNIFICANCE STATEMENT To our knowledge, this is the first study to determine the contribution of aldo-keto reductase (AKR)-1C1, AKR1C2, AKR1C3, AKR1C4, carbonyl reductase 1, and 11b-hydroxysteroid dehydrogenase type 1 to drug reductions in the human liver by utilizing the relative e
Forensic sample screening is important for establishing an effective DNA typing workflow. The detection of sexspecific markers in forensic samples highlights the necessity for further analysis. Y-chromosome DNA can co...
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Forensic sample screening is important for establishing an effective DNA typing workflow. The detection of sexspecific markers in forensic samples highlights the necessity for further analysis. Y-chromosome DNA can confirm male contributions, but female contributions are difficult to confirm using DNA-based methods. To address this, we developed a colorimetric reverse transcription loop-mediated isothermal amplification (RTLAMP) assay targeting the long non-coding RNA X-inactive specific transcript (XIST) to screen female samples. Operating at 65 degrees C for 30 min, the assay yielded results discernible from the color change of the pH indicator dye. The assay showed a detection limit of approximately 0.5 mu L of blood. The assay also detected XIST RNA in mixed body fluids and mock samples, indicating its potential applicability to casework samples. Taken together, our assay provides a rapid and simple strategy for screening female samples.
Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, t...
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Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 degrees C for 20 min;the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 degrees C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 mu L of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.
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