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Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys. Environmental DNA (eDNA) metabarcoding has enormous potential for community biodiversity assessment but the detection sensitivity of this tool for single species, particularly rare species, within communities is relatively unexplored. We compared targeted real‐time quantitative PCR (qPCR) and eDNA metabarcoding for great crested newt (Triturus cristatus) detection across 532 ponds in the UK and found metabarcoding was less sensitive than qPCR depending on detection thresholds applied. However, sequence read count was correlated with number of positive qPCR replicates, cost of both methods was comparable, and metabarcoding revealed a variety of aquatic and terrestrial fauna alongside great crested newt, thus metabarcoding can be used for initial survey of water bodies to better inform species‐specific survey.

Purpose To identify potential molecular targets for lung cancer intervention and diagnosis, we analyzed the differential miRNA expression of peripheral blood between lung cancer patients and healthy controls. Methods Three pairs of cases’ and controls’ peripheral blood samples were evaluated for miRNA expression by microarray. 12 miRNAs were selected for RT‐PCR validation and target genes prediction. In addition, 4 miRNAs were selected for future validation by RT‐PCR in a large sample of 145 cases and 55 frequency‐matched healthy controls. Results A total of 338 differentially expressed miRNAs were screened and identified by microarray. According to the fold changes, the top ten upregulated miRNAs were hsa‐miR‐124‐3p, hsa‐miR‐379‐5p, hsa‐miR‐3655, hsa‐miR‐450b‐5p, hsa‐miR‐29a‐5p, hsa‐miR‐200a‐3p, hsa‐miR‐542‐3p, hsa‐miR‐138‐5p, hsa‐miR‐219a‐2‐3p, and hsa‐miR‐4701‐3p, and the top ten downregulated miRNAs were hsa‐miR‐34c‐5p, hsa‐miR‐135a‐5p, hsa‐miR‐132‐3p, hsa‐miR‐3178, hsa‐miR‐4449, hsa‐miR‐4999‐3p, hsa‐miR‐1246, hsa‐miR‐4424, hsa‐miR‐1252‐5p, and hsa‐miR‐24‐2‐5p. RT‐PCR verification of the 12 miRNAs revealed that 5 of 8 upregulated miRNAs, 2 of 4 downregulated miRNAs showed a significant difference between the cases and controls (P < .05). A large number of target genes and their functional set showed overlapping among the 453 predicted target genes of the 12 miRNAs (P < .01). RT‐PCR in the large sample confirmed the significant differential expression level of hsa‐miR‐29a‐5p, hsa‐miR‐135a‐5p, hsa‐miR‐542‐3p, and hsa‐miR‐4491 between cases and controls (P < .05), and three of these microRNA, except hsa‐miR‐29a‐5p, were significant after Bonferroni correction for adjustment of multiple comparisons. Conclusion There was a significant difference in miRNAs expression in the peripheral blood between lung cancer patients and healthy controls, and 4 miRNAs were validated by a large‐size sample.

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

Background DNA double‐strand breaks (DSBs) are the most lethal and dangerous type of lesions with significant implications for both cellular function and organismal health. The number of DSBs (NDSBs) across the genome reflects DNA damage severity. However, current quantification methods mainly rely on next‐generation sequencing, which is laborious and expensive. This study aims to provide a simple, low‐cost, and high‐throughput standard curve‐based method for quantifying genome‐wide DSBs. Method Genomic DNA from human, mouse, Arabidopsis, Saccharomyces cerevisiae, and Escherichia coli was digested by seven blunt‐end restriction enzymes to generate DSB standards. Theoretical NDSBs for each standard were calculated based on restriction site frequency. Ligation‐mediated quantitative PCR (LM‐qPCR) was performed to obtain the Ct values, which were plotted against log‐transformed NDSBs to construct standard curves. Method reliability was assessed by comparing results with neutral single‐cell gel electrophoresis and γ‐H2AX flow cytometry. Results All genomes were successfully digested by seven blunt‐end restriction enzymes to produce standard DSB fragments. Standard curves demonstrated high linearity (R2 > 0.95), with intra‐ and inter‐assay coefficients of variation of 1.101% and 2.528%, respectively. The detection limit was below 100 DSBs. Quantification results strongly correlated with traditional DSB detection methods (|r| > 0.9). Conclusion This standard curve‐based method enables accurate, reproducible quantification of genome‐wide DSBs in various organisms. It is simple, low‐cost, and easily standardized, offering a promising tool for applications in genotoxicity testing, environmental exposure monitoring, and DNA damage research. This study presents a simple and cost‐effective method for quantifying DNA double‐strand breaks (DSBs) using standard curves based on Ct values from LM‐qPCR and the theoretical number of DSBs from enzyme‐digested DNA standards. Its simplicity, affordability, and high‐throughput capability make it a valuable tool for genotoxicity testing, environmental monitoring, and DNA damage studies across diverse organisms.

In recent years, the analysis of environmental DNA (eDNA) has significantly improved, allowing for high‐resolution species identification and possible biomass quantification from water samples. Fisheries management typically requires monitoring of catches, including precise information about bycatch quantities to make sound assessments of exploitation rates. Bycatch assessment is particularly challenging in large catches (>500 T), and the current practice of visual assessment of subsampled catches is time‐consuming, requires extensive labor, and often has low precision. We explored the feasibility for applying eDNA‐based methods for studying catch composition using the pelagic North Sea herring fishery with bycatch of mackerel as a case. First, we experimentally simulate a series of catches using a range of herring and mackerel weight proportions to establish relationships under real fisheries scenarios. The relationship is subsequently used to estimate the biomass of mackerel bycatch from eDNA from three herring catches, by sampling and comparing processing water both onboard ships and at the processing factory. All samples are analyzed using species‐specific quantitative PCR (qPCR). The experiments reveled a strong correlation between DNA and weight fractions characterized by a constant overrepresentation of mackerel DNA compared to expected mackerel weight. We found that eDNA‐based and visual methods applied to the same landing reflect the within catch variability in species composition alike, however, the methods can show disparity in total estimates of mackerel biomass. Accounting for haul mixing within total landed catches increases the precision of the factory and ship eDNA‐based estimates for the same catch. We show that eDNA‐based bycatch estimates provide coherent quantitative data, and likely improve quality and reduce costs of collecting fisheries‐dependent data and thereby contribute to securing sustainable fisheries. Our study paves the way for a reliable, eDNA based approach for quantitative estimation of bycatch in fisheries where we first used an experimental approach to elucidate the eDNA to biomass relationship in mixtures of two commercially valuable species, herring and mackerel, simulating realistic scenarios under commercial fishing. Subsequently, the eDNA‐to‐biomass relationship was used to estimate the biomass of bycatch mackerel in three real herring catches and compared with traditionally applied visual assessments. The weights of mackerel estimated from eDNA samples were comparable to estimates derived from routinely used visual based metrics, moreover the eDNA‐based method stood out in precision of the estimates.

Recent studies have revealed the significant role of SMYD3 and EZH2 genes in the development and aggressiveness of numerous types of malignant tumor. Therefore, the present study aimed to investigate the expression of SMYD3 and EZH2 in papillary thyroid cancer, and to determine the correlation between the expression of these genes and clinical characteristics. Resected thyroid tissue samples from 62 patients with papillary thyroid cancer were investigated. Thyroid tissue derived from the healthy regions of removed nodular goiters from 30 patients served as the control group. Reverse transcription-quantitative PCR analysis was employed to detect relative mRNA expression levels. Primer sequences and TaqMan® hydrolysis probe positions for EZH2 and SMYD3 were determined using the Roche Universal ProbeLibrary Assay Design Center version 2.50. EZH2 expression was detected in all thyroid cancer samples and in 83.3% of benign lesions. Notably, EZH2 was revealed to be upregulated in thyroid cancer tissues compared with control tissues (P=0.0002). EZH2 expression was positively correlated with tumor stage (P<0.0001; r=0.504), and multiple comparison analysis revealed that the highest expression of EZH2 was detected in samples staged pT4 (P=0.0001). SMYD3 expression was detected in all thyroid cancer samples and in 96.7% of healthy thyroid tissues; notably, the expression levels were similar in both groups. In addition, there was no correlation between SMYD3 expression and the aggressiveness of papillary thyroid cancer. In conclusion, overexpression of the EZH2 gene may be associated with the development of papillary thyroid cancer and EZH2 may be a potential therapeutic target in papillary thyroid cancer.

Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost–benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.

Premise To genetically discriminate subspecies of the common reed (Phragmites australis), we developed real‐time quantitative (qPCR) assays for identifying P. australis subsp. americanus, P. australis subsp. australis, and P. australis subsp. berlandieri. Methods and Results Utilizing study‐generated chloroplast DNA sequences, we developed three novel qPCR assays. Assays were verified on individuals of each subspecies and against two non‐target species, Arundo donax and Phalaris arundinacea. One assay amplifies only P. australis subsp. americanus, one amplifies P. australis subsp. australis and/or P. australis subsp. berlandieri, and one amplifies P. australis subsp. americanus and/or P. australis subsp. australis. This protocol enhances currently available rapid identification methods by providing genetic discrimination of all three subspecies. Conclusions The newly developed assays were validated using P. australis samples from across the United States. Application of these assays outside of this geographic range should be preceded by additional testing.

MicroRNA (miRNA/miR) 5′-isoforms (5′-isomiRs) differ from canonical sequences registered in the microRNA database in the length of their 5′ ends. The 'seed sequence' of miRNAs that bind to target mRNAs is 2-8 nucleotides from the 5′ end; thus, shifts at the 5′ end can cause a 'seed shift'. Accumulating data from miRNA deep sequencing have revealed that, in a substantial number of miRNAs, sequences corresponding to specific isomiRs, not the canonical form, are the most abundant. Studies have so far focused on circulating miRNAs as either markers or intercellular communication factors. miR-1246 is abundant in the serum and is a candidate diagnostic and prognostic marker for esophageal squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma, colorectal adenocarcinoma and non-small cell lung cancer (NSCLC). The present study analyzed the 5′-end of serum miR-1246 by fragment analysis and found that a 5′-isomiR, which is two bases shorter than the canonical sequence, was the most abundant sequence in patients with NSCLC as well as healthy donors. To quantify the 5′-isomiR, 5′-isomiR-specific primers based on primers for allele specific-PCR were used, primarily because commercially available methods for miRNA Reverse transcription-quantitative PCR cannot discriminate among sequences, especially those located at the 5′ end of miRNA. The total miR-1246 levels were significantly increased in patients with NSCLC; by contrast, the level of the canonical sequence was significantly decreased. Significant positive correlations were observed between the total miR-1246 levels and the 5′-isomiR levels, but not that of the canonical sequence. These results imply that the increase in levels of serum miR-1246 in patients with NSCLC depends on increase of the 5′-isomiR.

Blastocystis sp. is a single-celled parasite estimated to colonize the digestive tract of 1 to 2 billion people worldwide. Although it represents the most frequent intestinal protozoa in human stools, it remains still under-investigated in countries with a high risk of infection due to poor sanitary and hygiene conditions, such as in Africa. Therefore, the present study was carried out to determine the prevalence and subtype (ST) distribution of Blastocystis sp. in the Guinean population. For this purpose, fecal samples were collected from 500 individuals presenting or not digestive disorders in two hospitals of Conakry. Search for the parasite in stools was performed by real-time PCR targeting the small subunit rDNA gene followed by sequencing of the PCR products for subtyping of the isolates. A total of 390 participants (78.0%) was positive for Blastocystis sp. Five STs were identified in the Guinean cohort (ST1, ST2, ST3, ST4 and ST14) with varying frequency, ST3 being predominant. Among them, ST4 was found in only two patients confirming its global rarity in Africa whereas infections by ST14 were likely the result of zoonotic transmission from bovid. No significant association was detected between Blastocystis sp. colonization or ST distribution and the symptomatic status of Guinean subjects or the presence of digestive symptoms. In contrast, drilling water consumption represented a significant risk factor for infection by Blastocystis sp. Predominance of ST3 coupled with its low intra-ST diversity strongly suggested large-scale human-to-human transmission of this ST within this cohort. In parallel, the highest intra-ST diversity of ST1 and ST2 was likely correlated with various potential sources of infection in addition to anthroponotic transmission. These findings highlighted the active circulation of the parasite in Guinea as reported in some low-income African countries and the necessity to implement prevention and control measures in order to limit the circulation of this parasite in this endemic geographical area.