Circulating MicroRNAs Indicative of Sex and Stress in the European Seabass (Dicentrarchus labrax): Toward the Identification of New Biomarkers

MicroRNAs (miRNAs) constitute a new category of biomarkers. Studies on miRNAs in non-mammalian species have drastically increased in the last few years. Here, we explored the use of miRNAs as potential, poorly invasive markers, to identify sex and characterize acute stress in fish. The European seabass (Dicentrarchus labrax) was chosen as a model because of its rapid response to stress and its specific sex determination system, devoid of sexual chromosomes. We performed a small RNA-sequencing analysis in the blood plasma of male and female European seabass (mature and immature) as well as in the blood plasma of juveniles submitted to an acute stress and sampled throughout the recovery period (at 0 h, 0.5 h, 1.5 h and 6 h). In immature individuals, both miR-1388-3p and miR-7132a-5p were up-regulated in females, while miR-499a-5p was more abundant in males. However, no miRNAs were found to be differentially expressed between sexes in the blood plasma of mature individuals. For the acute stress analysis, five miRNAs (miR-155-5p, miR-200a-3p, miR-205-1-5p, miR-143-3p, and miR-223-3p) followed cortisol production over time. All miRNAs identified were tested and validated by RT-qPCR on sequenced samples. A complementary analysis on the 3′UTR sequences of the European seabass allowed to predict potential mRNA targets, some of them being particularly relevant regarding stress regulation, e.g., the glucocorticoid receptor 1 and the mineralocorticoid receptor. The present study provides new avenues and recommendations on the use of miRNAs as biomarkers of sex or stress of the European seabass, with potential application on other fish species

Intergenerational effects on fertility in male and female mice after chronic exposure to environmental doses of NSAIDs and 17α-ethinylestradiol mixtures

Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinylestradiol (EE2) are extensively used in human and veterinary medicine. Due to their partial removal by wastewater treatment plants, they are frequent environmental contaminants, particularly in drinking water. Here, we investigated the adverse outcomes of chronic exposure to mixtures of NSAIDs (ibuprofen, 2hydroxy-ibuprofen, diclofenac) and EE2 at two environmentally relevant doses in drinking water, on the reproductive organ development and fertility in F1-exposed male and female mice and in their F2 offspring. In male and female F1 mice, which were exposed to these mixtures, reproductive organ maturation, estrous cyclicity, and spermiogenesis were altered. These defects were observed also in F2 animals, in addition to some specific sperm parameter alterations in F2 males. Transcriptomic analysis revealed significant changes in gene expression patterns and associated pathways implicated in testis and ovarian physiology. Chronic exposure of mice to NSAID and EE2 mixtures at environmental doses intergenerationally affected male and female fertility (i.e. total number of pups and time between litters). Our study provides new insights into the adverse effects of these pharmaceuticals on the reproductive health and will facilitate the implementation of a future regulatory environmental risk assessment of NSAIDs and EE2 for human health

Imaging modalities to inform the detection and diagnosis of early caries

BACKGROUND: The detection and diagnosis of caries at the earliest opportunity is fundamental to the preservation of tooth tissue and maintenance of oral health. Radiographs have traditionally been used to supplement the conventional visual‐tactile clinical examination. Accurate, timely detection and diagnosis of early signs of disease could afford patients the opportunity of less invasive treatment with less destruction of tooth tissue, reduce the need for treatment with aerosol‐generating procedures, and potentially result in a reduced cost of care to the patient and to healthcare services. OBJECTIVES: To determine the diagnostic accuracy of different dental imaging methods to inform the detection and diagnosis of non‐cavitated enamel only coronal dental caries. SEARCH METHODS: Cochrane Oral Health's Information Specialist undertook a search of the following databases: MEDLINE Ovid (1946 to 31 December 2018); Embase Ovid (1980 to 31 December 2018); US National Institutes of Health Ongoing Trials Register (ClinicalTrials.gov, to 31 December 2018); and the World Health Organization International Clinical Trials Registry Platform (to 31 December 2018). We studied reference lists as well as published systematic review articles. SELECTION CRITERIA: We included diagnostic accuracy study designs that compared a dental imaging method with a reference standard (histology, excavation, enhanced visual examination), studies that evaluated the diagnostic accuracy of single index tests, and studies that directly compared two or more index tests. Studies reporting at both the patient or tooth surface level were included. In vitro and in vivo studies were eligible for inclusion. Studies that explicitly recruited participants with more advanced lesions that were obviously into dentine or frankly cavitated were excluded. We also excluded studies that artificially created carious lesions and those that used an index test during the excavation of dental caries to ascertain the optimum depth of excavation. DATA COLLECTION AND ANALYSIS: Two review authors extracted data independently and in duplicate using a standardised data extraction form and quality assessment based on QUADAS‐2 specific to the clinical context. Estimates of diagnostic accuracy were determined using the bivariate hierarchical method to produce summary points of sensitivity and specificity with 95% confidence regions. Comparative accuracy of different radiograph methods was conducted based on indirect and direct comparisons between methods. Potential sources of heterogeneity were pre‐specified and explored visually and more formally through meta‐regression. MAIN RESULTS: We included 104 datasets from 77 studies reporting a total of 15,518 tooth sites or surfaces. The most frequently reported imaging methods were analogue radiographs (55 datasets from 51 studies) and digital radiographs (42 datasets from 40 studies) followed by cone beam computed tomography (CBCT) (7 datasets from 7 studies). Only 17 studies were of an in vivo study design, carried out in a clinical setting. No studies were considered to be at low risk of bias across all four domains but 16 studies were judged to have low concern for applicability across all domains. The patient selection domain had the largest number of studies judged to be at high risk of bias (43 studies); the index test, reference standard, and flow and timing domains were judged to be at high risk of bias in 30, 12, and 7 studies respectively. Studies were synthesised using a hierarchical bivariate method for meta‐analysis. There was substantial variability in the results of the individual studies, with sensitivities that ranged from 0 to 0.96 and specificities from 0 to 1.00. For all imaging methods the estimated summary sensitivity and specificity point was 0.47 (95% confidence interval (CI) 0.40 to 0.53) and 0.88 (95% CI 0.84 to 0.92), respectively. In a cohort of 1000 tooth surfaces with a prevalence of enamel caries of 63%, this would result in 337 tooth surfaces being classified as disease free when enamel caries was truly present (false negatives), and 43 tooth surfaces being classified as diseased in the absence of enamel caries (false positives). Meta‐regression indicated that measures of accuracy differed according to the imaging method (Chi(2)(4) = 32.44, P < 0.001), with the highest sensitivity observed for CBCT, and the highest specificity observed for analogue radiographs. None of the specified potential sources of heterogeneity were able to explain the variability in results. No studies included restored teeth in their sample or reported the inclusion of sealants. We rated the certainty of the evidence as low for sensitivity and specificity and downgraded two levels in total for risk of bias due to limitations in the design and conduct of the included studies, indirectness arising from the in vitro studies, and the observed inconsistency of the results. AUTHORS' CONCLUSIONS: The design and conduct of studies to determine the diagnostic accuracy of methods to detect and diagnose caries in situ are particularly challenging. Low‐certainty evidence suggests that imaging for the detection or diagnosis of early caries may have poor sensitivity but acceptable specificity, resulting in a relatively high number of false‐negative results with the potential for early disease to progress. If left untreated, the opportunity to provide professional or self‐care practices to arrest or reverse early caries lesions will be missed. The specificity of lesion detection is however relatively high, and one could argue that initiation of non‐invasive management (such as the use of topical fluoride), is probably of low risk. CBCT showed superior sensitivity to analogue or digital radiographs but has very limited applicability to the general dental practitioner. However, given the high‐radiation dose, and potential for caries‐like artefacts from existing restorations, its use cannot be justified in routine caries detection. Nonetheless, if early incidental carious lesions are detected in CBCT scans taken for other purposes, these should be reported. CBCT has the potential to be used as a reference standard in diagnostic studies of this type. Despite the robust methodology applied in this comprehensive review, the results should be interpreted with some caution due to shortcomings in the design and execution of many of the included studies. Future research should evaluate the comparative accuracy of different methods, be undertaken in a clinical setting, and focus on minimising bias arising from the use of imperfect reference standards in clinical studies