Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.
33Species-level genetic characterization of complex bacterial communities has important 34 clinical applications. In the present study, we assessed the performance of full-length 35 16S rRNA gene analysis of human gut microbiota using the nanopore long-read 36 sequencer MinION™. A new strategy for library construction with an optimized primer 37 set overcame PCR-associated bias and produced accurate taxonomic classifications of a 38 broad range of bacterial species. Our present microbiome study, comparing the 39 discriminatory power of full-length and short-read sequencing, clearly illustrated the 40 analytical advantage of sequencing the full-length 16S rRNA gene, which provided 41 higher species-level resolution and accuracy. 42 43 Keywords: 44 16S rRNA, gut microbiota, metagenome, MinION™, nanopore sequencing 45 46 4 Background 47 Recent advances in DNA sequencing technology have had a revolutionary impact on 48 clinical microbiology [1]. Next-generation sequencing (NGS) technology enables 49 parallel sequencing of DNA on a massive scale to generate vast quantities of accurate 50 data. NGS platforms are now increasingly used in the field of clinical research [2]. 51 Metagenomic sequencing offers numerous advantages over traditional culture-based 52 techniques that have long been the standard test for detecting pathogenic bacteria. This 53 method is particularly useful for characterizing uncultivable bacteria and novel 54 pathogens [3].55 Among the metagenomic sequencing strategies, amplicon sequencing of the 16S 56 ribosomal RNA (rRNA) gene has proven to be a reliable and efficient option for 57 taxonomic classification [4, 5]. The bacterial 16S rRNA gene contains nine variable 58 regions (V1 to V9) that are separated by highly conserved sequences across different 59 taxa. For bacterial identification, the 16S rRNA gene is first amplified by polymerase 60 chain reaction (PCR) with primers annealing to conserved regions and then sequenced.61 The sequencing data are subjected to bioinformatic analysis in which the variable 62 regions are used to discriminate between bacterial taxa [6]. 63 Since the conventional parallel-type short-read sequencer cannot yield reads covering 64 the full length of the 16S rRNA gene [7], several regions of it have been targeted for 65 sequencing, which often causes ambiguity in taxonomic classification [8]. New 66 sequencing platforms have overcome these technical restrictions, particularly those 67 affecting read length. A prime example is the MinION™ sequencer from Oxford 68Nanopore Technologies, which is capable of producing long sequences with no 69 theoretical read length limit [9][10][11]. MinION™ sequencing targets the entire 16S rRNA 70 gene, allowing the identification of bacteria with more accuracy and sensitivity [12, 13]. 71Furthermore, MinION™ produces sequencing data in real time, which reduces 72 turnaround time for data processing [14, 15]. 73 5Given these features of MinION™ sequencing, we had previously conducted full-length 74 16S amplicon sequencing analyse...
Endometrial stromal cells differentiate into decidual cells through the process of decidualization. This differentiation is critical for embryo implantation and the successful establishment of pregnancy. Recent epidemiological studies have suggested that thyroid hormone is important in the endometrium during implantation, and it is commonly believed that thyroid hormone is essential for proper development, differentiation, growth, and metabolism. This study aimed to investigate the impact of thyroid hormone on decidualization in human endometrial stromal cells (hESCs) and define its physiological roles in vitro by gene targeting. To identify the expression patterns of thyroid hormone, we performed gene expression profiling of hESCs during decidualization after treating them with the thyroid hormone levothyroxine (LT4). A major increase in decidual response was observed after combined treatment with ovarian steroid hormones and thyroid hormone. Moreover, LT4 treatment also affected the regulation of many transcription factors important for decidualization. We found that type 3 deiodinase, which is particularly important in fetal and placental tissues, was upregulated during decidualization in the presence of thyroid hormone. Further, it was observed that progesterone receptor, an ovarian steroid hormone receptor, was involved in thyroid hormone–induced decidualization. In the absence of thyroid hormone receptor (TR), due to the simultaneous silencing of TRα and TRβ, thyroid hormone expression was unchanged during decidualization. In summary, we demonstrated that thyroid hormone is essential for decidualization in the endometrium. This is the first in vitro study to find impaired decidualization as a possible cause of infertility in subclinical hypothyroidism (SCH) patients.
The study aimed to elucidate the glycolytic metabolism of human endometrial stromal cells (hESCs) in hypoxic environment. Main methods: The hESCs were cultured in hypoxic environment, and their metabolic pathways were analyzed using metabolomics. We assessed glucose uptake using 2-deoxyglucose (2-DG) assay. The expression of glucose transporters (GLUTs) required for glucose uptake was determined using real-time quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, we knocked down GLUT1 and examined the uptake of 2-DG. Key findings: Under hypoxia, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate were significantly elevated in hESCs (P < 0.05). This finding indicated enhancement in glycolysis. The volume of glucose uptake increased significantly under hypoxia (P < 0.05). Hypoxia simultaneously induced the expression of GLUT1 and GLUT3 mRNA (P < 0.05) and attenuated the expression of GLUT8 (P < 0.05). Glucose uptake was significantly inhibited upon knockdown of GLUT1 (P < 0.0001). Significance: These results demonstrated a very important role of glucose transport under hypoxia. Also, hESCs utilize glycolysis to adapt to hypoxic conditions that could occur in menstrual and implantation period. These findings pave the way to study implantation failure and tumors originating from the endometrium.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.