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To improve our ability to extrapolate preclinical toxicity to humans, there is a need to understand and quantify the concordance of adverse events (AEs) between animal models and clinical studies. In the present work, we discovered 3011 statistically significant associations between preclinical and clinical AEs caused by drugs reported in the PharmaPendium database of which 2952 were new associations between toxicities encoded by different Medical Dictionary for Regulatory Activities terms across species. To find plausible and testable candidate off-target drug activities for the derived associations, we investigated the genetic overlap between the genes linked to both a preclinical and a clinical AE and the protein targets found to interact with one or more drugs causing both AEs. We discuss three associations from the analysis in more detail for which novel candidate off-target drug activities could be identified, namely, the association of preclinical mutagenicity readouts with clinical teratospermia and ovarian failure, the association of preclinical reflexes abnormal with clinical poor-quality sleep, and the association of preclinical psychomotor hyperactivity with clinical drug withdrawal syndrome. Our analysis successfully identified a total of 77% of known safety targets currently tested in in vitro screening panels plus an additional 431 genes which were proposed for investigation as future safety targets for different clinical toxicities. This work provides new translational toxicity relationships beyond AE term-matching, the results of which can be used for risk profiling of future new chemical entities for clinical studies and for the development of future in vitro safety panels.

Funder: Unilever, Estimation of points of departure (PoDs) from high-throughput transcriptomic data (HTTr) represents a key step in the development of next-generation risk assessment (NGRA). Current approaches mainly rely on single key gene targets, which are constrained by the information currently available in the knowledge base and make interpretation challenging as scientists need to interpret PoDs for thousands of genes or hundreds of pathways. In this work, we aimed to address these issues by developing a computational workflow to investigate the pathway concentration-response relationships in a way that is not fully constrained by known biology and also facilitates interpretation. We employed the Pathway-Level Information ExtractoR (PLIER) to identify latent variables (LVs) describing biological activity and then investigated in vitro LVs' concentration-response relationships using the ToxCast pipeline. We applied this methodology to a published transcriptomic concentration-response data set for 44 chemicals in MCF-7 cells and showed that our workflow can capture known biological activity and discriminate between estrogenic and antiestrogenic compounds as well as activity not aligning with the existing knowledge base, which may be relevant in a risk assessment scenario. Moreover, we were able to identify the known estrogen activity in compounds that are not well-established ER agonists/antagonists supporting the use of the workflow in read-across. Next, we transferred its application to chemical compounds tested in HepG2, HepaRG, and MCF-7 cells and showed that PoD estimates are in strong agreement with those estimated using a recently developed Bayesian approach (cor = 0.89) and in weak agreement with those estimated using a well-established approach such as BMDExpress2 (cor = 0.57). These results demonstrate the effectiveness of using PLIER in a concentration-response scenario to investigate pathway activity in a way that is not fully constrained by the knowledge base and to ease the biological interpretation and support the development of an NGRA framework with the ability to improve current risk assessment strategies for chemicals using new approach methodologies.

Supplementary data related to this article: Supplementary material, available at https://ars.els-cdn.com/content/image/1-s2.0-S0048969722047647-mmc1.docx (Word document35, 5KB)); Supplementary Data 1. These are the chemicals along with their properties and the calculated doses given for the C. elegans (Caenorhabditis elegans), available at https://ars.els-cdn.com/content/image/1-s2.0-S0048969722047647-mmc2.xls (spreadsheet, 68KB); Supplementary Data 2. These are the chemicals and the doses given for the trout gill cell line (Rtgill-WT1). The codes here relate to the same codes on the microarray data, available at https://ars.els-cdn.com/content/image/1-s2.0-S0048969722047647-mmc3.xlsx (spreadsheet; 11KB); Supplementary Data 3. These are the probes found to be significantly different between exposed and unexposed C. elegans for each of the narcotic chemicals tested, available at https://ars.els-cdn.com/content/image/1-s2.0-S0048969722047647-mmc4.xlsx (spreadsheet, 363KB); Supplementary Data 4. The file provide the metabolite composition of the three clusters identified by running a metabolic-based predictive model, available at https://ars.els-cdn.com/content/image/1-s2.0-S0048969722047647-mmc5.xlsx (spreadsheet, 10KB). Copyright © 2022 The Authors. With the large numbers of man-made chemicals produced and released in the environment, there is a need to provide assessments on their potential effects on environmental safety and human health. Current regulatory frameworks rely on a mix of both hazard and risk-based approaches to make safety decisions, but the large number of chemicals in commerce combined with an increased need to conduct assessments in the absence of animal testing makes this increasingly challenging. This challenge is catalysing the use of more mechanistic knowledge in safety assessment from both in silico and in vitro approaches in the hope that this will increase confidence in being able to identify modes of action (MoA) for the chemicals in question. Here we approach this challenge by testing whether a functional genomics approach in C. elegans and in a fish cell line can identify molecular mechanisms underlying the effects of narcotics, and the effects of more specific acting toxicants. We show that narcosis affects the expression of neuronal genes associated with CNS function in C. elegans and in a fish cell line. Overall, we believe that our study provides an important step in developing mechanistically relevant biomarkers which can be used to screen for hazards, and which prevent the need for repeated animal or cross-species comparisons for each new chemical. Unilever Ltd.

Effective stock assessment relies on the evaluation of the reproductive potential of a population, but this is often achieved using fast, inexpensive, and inaccurate macroscopic indicators of gonadal maturity. Indeed, this approach suffers from high error rates as a result of misclassification and operator errors. Although histology is considered the gold standard to assign reproductive stages, it represents a time‐consuming and expensive method. Therefore, cost‐effective approaches are currently needed to assess the species‐specific reproductive potential and define the correct size at first maturity (L₅₀). In the present study a histology‐calibrated method based on the gonadal index (GI) was developed to assign maturity stages to the overfished Mediterranean swordfish (Xiphias gladius), supported by histological classification of gonadal development, an estimation of L₅₀, and an analysis of the reproductive output of this species. The performance of this new method had an overall agreement of 90% with histological data, and the L₅₀ calculated by the GI‐based method compared with histology analysis was not statistically different (p > 0.05). Moreover, in order to apply this method without bias, a thorough validation of the relationship between length and gonad weight was carried out and the suitability of the GI to compare individuals of different size and maturity stages was confirmed. The present findings provide a comprehensive picture of the patterns characterizing Mediterranean swordfish reproduction and a promising method to assign maturity stages based on a macroscopic indicator. This method has the potential to be extensively applied during routine fish sampling activities in the field because of its simple requirements.

Simple Summary The Atlantic bluefin tuna Thunnus thynnus is a species characterized by complex trans-oceanic migrations linked to size, which rely on the delicate trade-off between somatic growth and reproduction before and during the migratory movements to reach spawning grounds. Therefore, understanding the processes that drive reproduction and elucidating its age-related regulation is essential in the context of sustainable fishery management. In this study, carried out in the Mediterranean Sea, older bluefin tuna females were found to have greater reproductive performances than younger females according to a molecular biology approach (i.e., gene expression), a result that likely mirrors a better physical condition, different habitat usage or migratory behaviour. This result highlights the importance of preserving large females for their major reproductive contribution at a stock level. Furthermore, the gonad-specific mir-202, which belongs to a class of non-coding RNA, called miRNA, that regulate the post-transcription of protein-coding genes, was identified as a potential candidate to play a role in egg quality and quantity (i.e., fecundity) during ovarian maturation through age- or stage-dependent reproductive processes. Overall, the present study contributes to improve the sustainability of the Atlantic bluefin tuna fishery in the Mediterranean Sea. Abstract In the Mediterranean Sea, a demographic substructure of the Atlantic bluefin tuna Thunnus thynnus has emerged over the last decade, with old and young individuals exhibiting different horizontal movements and spatial–temporal patterns of gonad maturation. In the present study, histology and molecular reproductive markers were integrated with the gonad-specific mir-202 gene expression and ovarian localization to provide a comprehensive picture of the reproductive performances in young and old females and investigate the role played by the mir-202 during gonadal maturation. During the reproductive period, old females (>100 kg; 194.6 ± 33.9 cm straight fork length; 11.3 ± 2.7 years old) were found to have greater reproductive performances than younger females (

The agricultural sector is required to produce food at the same pace as population growth, while accounting for pollution and costs. For this reason, conservative agricultural practices have been employed worldwide. Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy has the ability to provide a snapshot of the macromolecular composition of a sample in a timely and cost-effective way and it has been widely applied in the field of agriculture to assess food quality. The aim of this study was to exploit ATR-FTIR spectroscopy to assess the impact of different soil tillage methods (conventional tillage, CT, minimum tillage, MT, and no tillage, NT) and nitrogen fertilization levels (0, 90 and 180 kg N ha−1) on the macromolecular composition of leaves and caryopses of durum wheat (Triticum turgidum subsp. durum). The analysis of the spectral data revealed that the quality of durum wheat, in terms of protein content, grown on soil with no tillage was not reduced. Indeed, with regards to caryopses, the different tillage methods influenced only the lipid and hemicellulose content, whereas the macromolecular composition of leaves was sensitive to tillage methods mostly during the early stage of growth. Moreover, no relevant effects were found in leaves and caryopses when different fertilizer concentrations were used. These results provide important knowledge supporting the adoption of both no-tillage soil treatments and reduced fertilization dosage for the development of durum wheat management strategies and support the use of spectroscopy for conservative agriculture practices.

The use of probiotics has been recently considered a novel therapeutic strategy to prevent pathologies such as obesity; however, the specific mechanisms of action by which probiotics exert their beneficial effects on metabolic health remain unclear. The aim of the present study was to investigate the short-term effects of a probiotic Lactobacillus rhamnosus supplementation (PROB) on appetite regulation, growth-related markers, and microbiota diversity in zebrafish (Danio rerio) larvae, compared to a group subjected to a constant darkness photoperiod (DARK), as well as to evaluate the effects of both treatments on melatonin receptors' expression. After a 24 h treatment, both PROB and DARK conditions caused a significant increase in leptin a expression. Moreover, mRNA abundances of leptin b and proopiomelanocortin a were elevated in the PROB group, and DARK showed a similar tendency, supporting a negative regulation of appetite markers by the treatments. Moreover, both PROB and DARK also enhanced the abundances of melatonin receptors transcript (melatonin receptor 1 ba and bb) and protein (melatonin receptor 1) suggesting a potential involvement of melatonin in mediating these effects. Nevertheless, treatments did not exhibit a significant effect on the expression of most of the growth hormone/insulin-like growth factor axis genes evaluated. Finally, only the DARK condition significantly modulated gut microbiota diversity at such short time, altogether highlighting the rapid effects of this probiotic on modulating appetite regulatory and melatonin receptors' expression, without a concomitant variation of gut microbiota.

Over the last two decades, many efforts have been invested in attempting to close the life cycle of the iconic Atlantic bluefin tuna (Thunnus thynnus) and develop a true aquaculture-based market. However, the limited molecular resources nowadays available represent a clear limitation towards the domestication of this species. To fill such a gap of knowledge, we assembled and characterized a de novo larval transcriptome by taking advantage of publicly available databases with the final goal of better understanding the larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3,061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KAAS annotation server. By comparing our data with a set of representative fish species proteomes, it was found that 78.4% of the tuna transcripts were successfully included in orthologous groups. Codon usage bias was identified for processes such as translation, peptide biosynthesis, muscle development and ion transport, supporting the idea of mechanisms at play in regulating stability and translation efficiency of transcripts belonging to key biological processes during the larval growth. The information generated by this study on the Atlantic bluefin tuna represent a relevant improvement of the transcriptomic resources available to the scientific community and lays the foundation for future works aimed at exploring in greater detail physiological responses at molecular level in different larval stages.

Handheld chlorophyll meters as Soil Plant Analysis Development (SPAD) have proven to be useful tools for rapid, no-destructive assessment of chlorophyll and nitrogen status in various crops. This method is used to diagnose the need of nitrogen fertilization to improve the efficiency of the agricultural system and to minimize nitrogen losses and deficiency. The objective of this study is to evaluate the effect of repeated conservative agriculture practices on the SPAD readings, leaves chlorophyll concentration and Nitrogen Nutrition Index (NNI) relationships in durum wheat under Mediterranean conditions. The experimental site is a part of a long-term-experiment established in 1994 and is still on-going where three tillage managements and three nitrogen fertilizer treatments were repeated in the same plots every year. We observed a linear relationship between the SPAD readings performed in the central and distal portion of the leaf (R2 = 0.96). In fertilized durum wheat, we found all positive exponential relationships between SPAD readings, chlorophyll leaves concentration (R2 = 0.85) and NNI (R2 = 0.89). In the unfertilized treatment, the SPAD has a good attitude to estimate leaves chlorophyll concentration (R2 = 0.74) and NNI (R2 = 0.77) only in crop grow a soil with relative high content of soil organic matter and nitrogen availability, as observed in the no tilled plots. The results show that the SPAD can be used for a correct assessment of chlorophyll and nitrogen status in durum wheat but also to evaluate indirectly the content of soil organic matter and nitrogen availability during different growth stages of the crop cycle.

Gut microbiota plays a fundamental role in maintaining host's health by controlling a wide range of physiological processes. Administration of probiotics and manipulation of photoperiod have been suggested as modulators of microbial composition and are currently undergoing an extensive research in aquaculture as a way to improve health and quality of harvested fish. However, our understanding regarding their effects on physiological processes is still limited. In the present study we investigated whether manipulation of photoperiod and/or probiotic administration was able to alter microbial composition in zebrafish larvae at hatching stage. Our findings show that probiotic does not elicit effects while photoperiod manipulation has a significant impact on microbiota composition. Moreover, we successfully predicted lipid biosynthesis and apoptosis to be modulated by microbial communities undergoing continuous darkness. Interestingly, expression levels of caspase 3 gene (casp3) and lipid-related genes (hnf4a, npc1l1, pparγ, srebf1, agpat4 and fitm2) were found to be significantly overexpressed in dark-exposed larvae, suggesting an increase in the occurrence of apoptotic processes and a lipid metabolism impairment, respectively (p

The Mediterranean swordfish (Xiphias gladius) has been recently classified as overfished and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish biology is needed. To date, few studies on swordfish have been performed and none of them has provided useful insights into the reproductive biology at molecular level. Here we set to characterise the molecular dynamics underlying ovarian maturation by employing a de novo transcriptome assembly approach. Differential gene expression analysis in mature and immature ovaries identified a number of differentially expressed genes associated with biological processes driving ovarian maturation. Focusing on ovarian steroidogenesis and vitellogenin uptake, we depict the molecular dynamics characterizing these processes while a phylogenetic analysis let us identify a candidate vitellogenin receptor. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.

Circadian rhythm is well known to play a pivotal role in reproduction but the presence of a gonadal circadian rhythm is opening a lot of questions about a local regulation of reproduction. In the present study, we first set to identify the key genes driving circadian rhythmicity from the gonadal transcriptome of the swordfish, a commercially relevant species undergoing overfishing, and we then investigated whether their transcriptional activity was influenced by the maturation stage. Finally, we explored whether seasonality had the ability to modulate the expression of these genes. Interestingly, we identified a wide number of circadian rhythm related genes in the transcriptome of the swordfish gonad including, among the others, clock, bmal1, cry2 and per2, which have been found to be differentially expressed between sexually immature and mature individuals sampled during the breeding season. This differential modulation was also found for melatonin biosynthesis genes (mel1b, asmt and tph1) as well as opsin genes (opsin4, tmt opsin, parapinopsin, VA opsin, rho and sws), known to be the primary receptors of light stimuli. These differences were not observed between mature and immature individuals when considering the non-breeding season suggesting that, despite the molecular machinery of mature gonads is able to respond to signals driving ovarian maturation, these signals are not present hence highlighting the potential role of seasonality in modulating the gonadal circadian rhythm. These results confirm the presence of a gonadal circadian rhythm in the swordfish and open new interesting questions about its role in driving puberty onset.

In recent years, decreases in fish populations have been attributed, in part, to the effect of environmental chemicals on ovarian development. To understand the underlying molecular events we developed a dynamic model of ovary development linking gene transcription to key physiological end points, such as gonadosomatic index (GSI), plasma levels of estradiol (E2) and vitellogenin (VTG), in largemouth bass ( Micropterus salmoides). We were able to identify specific clusters of genes, which are affected at different stages of ovarian development. A subnetwork was identified that closely linked gene expression and physiological end points and by interrogating the Comparative Toxicogenomic Database (CTD), quercetin and tretinoin (ATRA) were identified as two potential candidates that may perturb this system. Predictions were validated by investigation of reproductive associated transcripts using qPCR in ovary and in the liver of both male and female largemouth bass treated after a single injection of quercetin and tretinoin (10 and 100 μg/kg). Both compounds were found to significantly alter the expression of some of these genes. Our findings support the use of omics and online repositories for identification of novel, yet untested, compounds. This is the first study of a dynamic model that links gene expression patterns across stages of ovarian development.

Over the past 25 years, extensive research in vertebrate species has identified several genomic pathways altered by exposures to anthropogenic chemicals with hormone-like activity mediated by their interaction with nuclear receptors. In addition, many pollutants have been shown to interfere with non-genomic (non-classical) pathways, but this mechanism of endocrine disruption is still poorly understood. Recently, the number of publications describing the effects of Endocrine disrupting chemicals (EDCs) on fish reproduction, focusing on the deregulation of the hypothalamus-pituitary-gonadal axis as well as on gamete quality, significantly increased. Depending on their ability to mimic endogenous hormones, the may differently affect male or female reproductive physiology. Inhibition of gametogenesis, development of intersex gonads, alteration of the gonadosomatic index, and decreased fertility rate have been largely documented. In males, alterations of sperm density, motility, and fertility have been observed in several wild species. Similar detrimental effects were described in females, including negative outcomes on oocyte growth and maturation plus the occurrence of apoptotic/autophagic processes. These pathways may affect gamete viability considered as one of the major indicators of reproductive endocrine disruption. Pollutants act also at DNA level producing DNA mutations and changes in epigenetic pathways inducing specific mechanisms of toxicity and/or aberrant cellular responses that may affect subsequent generation(s) through the germline. In conclusion, this review summarizes the effects caused by EDC exposure on fish reproduction, focusing on gametogenesis, giving a general overview of the different aspects dealing with this issue, from morphological alteration, deregulation of steroidogenesis, hormonal synthesis, and occurrence of epigenetic process.

The acceleration of Digital Agriculture is evident through the increased adoption of digital technologies on farms including smart machines, sensors and cloud computing. In this paper we present the preliminary results of the research project funded by Università Politecnica delle Marche in 2018 “PFRLab: Setting of a precision farming robotic laboratory for cropping system sustainability and food safety and security”, which is still underway. In this context, as first result, an interdepartmental Research and Services Center called “Smart Farming” has been set up with the aim to strengthen multidisciplinary collaborations in the fields of Agriculture and Forestry, Geomatics, ICT and Robotics. Regarding field activities the SPAD 502 as well as Normalized Difference Vegetation Index (NDVI) provide a good estimate of the Chlorophylla+b content in durum wheat leaves so can be used to predict in a quickly and non-destructively way, the crop greenness status and to identify any nutritional deficiencies in real time. Future research activities are certainly needed to fully explore the potentialities of conservation agriculture and precision farming, and to drive the transition process from conventional agriculture to modern conservation agriculture and precision farming techniques. In-depth studies are planned on the combined effect of nitrogen fertilization and soil management on the main production variables of durum wheat in order to evaluate whether specific tools for precision agriculture applications can find significant diffusion even in Mediterranean cereal based cropping systems.