Microorganisms developing in the liner of the spent fuel pool (SFP) and the fuel transfer channel (FTC) of a Nuclear Power Plant (NPP) can form high radiation resistant biofilms and cause corrosion. Due to difficulties and limitations to obtain large samples from SFP and FTC, cotton swabs were used to collect the biofilm from the wall of these installations. Molecular characterization was performed using massively parallel sequencing to obtain a taxonomic and functional gene classification. Also, samples from the drainage system were evaluated because microorganisms may travel over the 12-meter column of the pool water of the Brazilian Nuclear Power Plant (Angra1), which has been functioning since 1985. Regardless of the treatment of the pool water, our data reveal the unexpected presence of Fungi (Basidiomycota and Ascomycota) as the main contaminators of the SFP and FTC. Ustilaginomycetes (Basidiomycota) was the major class contributor (70%) in the SFP and FTC reflecting the little diversity in these sites; nevertheless, Proteobacteria, Actinobacteria, Firmicutes (Bacilli) were present in small proportions. Mapping total reads against six fungal reference genomes indicate that there is, in fact, a high abundance of fungal sequences in samples collected from SFP and FTC. Analysis of the ribosomal internal transcribed spacer (ITS) 1 and 2 regions and the protein found in the mitochondria of eukaryotic cells, cytochrome b (cytb) grouped our sample fungi in the clade 7 as Ustilago and Pseudozyma. In contrast, in the drainage system, Alphaproteobacteria were present in high abundances (55%). The presence of Sphingopyxis, Mesorhizobium, Erythrobacter, Sphingomonas, Novosphingobium, Sphingobium, Chelativorans, Oceanicaulis, Acidovorax, and Cyanobacteria was observed. Based on genomic annotation data, the assessment of the biological function found a higher proportion of protein-coding sequences related to respiration and protein metabolism in SFP and FTC samples. The knowledge of this biological inventory present in the system may contribute to further studies of potential microorganisms that might be useful for bioremediation of nuclear waste.
Direct-acting antiviral (DAA)-based therapy is the new standard treatment for chronic hepatitis C virus (HCV) infection. However, protease inhibitor (PI)-resistant viral variants have been often described. This study aimed to examine HCV-NS3 protease variants at baseline and at 4 weeks under triple therapy. To this end, we analyzed the presence of variants in HCV-NS3 protease region from peripheral blood samples of 16 patients infected with HCV-1 at baseline and at 4 weeks of combined therapy with telaprevir, pegylated interferon, and ribavirin, using next-generation sequencing. Several variants with synonymous and non-synonymous amino acid substitutions were detected at both time points. Variants detected at low frequency corresponded to 74% (HCV-1a) and 35% (HCV-1b) of non-synonymous substitutions. We found nine PI-resistance-associated variants (V36A, T54S, V55I, Q80K, Q80R, V107I, I132V, D168E, M175L) in HCV-NS3 of 10 patients. There was no correspondence of resistance-associated variant profile between baseline and at 4 weeks. Moreover, these resistance variants at baseline and short-term treatment are not good predictors of outcome under triple therapy. Our study also shows a large number of others minor and major non-synonymous variants in HCV-NS3 early in telaprevir-based therapy that can be important for further drug resistance association studies with newly developed PI agents.
A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.
Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parallel sequencing (MPS) offers enhanced sensitivity and specificity for microbiological analyses. In addition, 16S rRNA target amplification followed by MPS facilitates the combined use of multiple markers/regions, better discrimination of sample background, and higher sample throughput. We designed a novel set of 16S rRNA gene primers for detection of bacterial species associated with clinical, bioweapon, and biohazards microorganisms via alignment of 364 sequences representing 19 bacterial species and strains relevant to medical and forensics applications. In silico results indicated that the hypervariable regions (V1V2), (V4V5), and (V6V7V8) support the resolution of a selected group of bacteria. Interspecies and intraspecies comparisons showed 74.23%–85.51% and 94.48%–99.98% sequencing variation among species and strains, respectively. Sequence reads from a simulated scenario of bacterial species mapped to each of the three hypervariable regions of the respective species with different affinities. The minimum limit of detection was achieved using two different MPS platforms. This protocol can be used to detect or monitor as low as 2,000 genome equivalents of bacterial species associated with clinical, bioweapon, and biohazard microorganisms and potentially can distinguish natural outbreaks of pathogenic microorganisms from those occurring by intentional release.
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