Determination of in vitro hepatotoxic potencies of a series of perfluoroalkyl substances (PFASs) based on gene expression changes in HepaRG liver cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse health effects, including hepatotoxicity, developmental toxicity, and immunotoxicity. The aim of the present work was to assess whether human HepaRG liver cells can be used to obtain insight into differences in hepatotoxic potencies of a series of PFASs. Therefore, the effects of 18 PFASs on cellular triglyceride accumulation (AdipoRed assay) and gene expression (DNA microarray for PFOS and RT-qPCR for all 18 PFASs) were studied in HepaRG cells. BMDExpress analysis of the PFOS microarray data indicated that various cellular processes were affected at the gene expression level. From these data, ten genes were selected to assess the concentration-effect relationship of all 18 PFASs using RT-qPCR analysis. The AdipoRed data and the RT-qPCR data were used for the derivation of in vitro relative potencies using PROAST analysis. In vitro relative potency factors (RPFs) could be obtained for 8 PFASs (including index chemical PFOA) based on the AdipoRed data, whereas for the selected genes, in vitro RPFs could be obtained for 11-18 PFASs (including index chemical PFOA). For the readout OAT5 expression, in vitro RPFs were obtained for all PFASs. In vitro RPFs were found to correlate in general well with each other (Spearman correlation) except for the PPAR target genes ANGPTL4 and PDK4. Comparison of in vitro RPFs with RPFs obtained from in vivo studies in rats indicate that best correlations (Spearman correlation) were obtained for in vitro RPFs based on OAT5 and CXCL10 expression changes and external in vivo RPFs. HFPO-TA was found to be the most potent PFAS tested, being around tenfold more potent than PFOA. Altogether, it may be concluded that the HepaRG model may provide relevant data to provide insight into which PFASs are relevant regarding their hepatotoxic effects and that it can be applied as a screening tool to prioritize other PFASs for further hazard and risk assessment.

Perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorononanoic acid (PFNA) increase triglyceride levels and decrease cholesterogenic gene expression in human HepaRG liver cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent in the environment, food chain, and humans. Epidemiological studies have shown a positive association between serum levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), and increased serum cholesterol and, in some cases, also triglyceride levels. However, causality has been questioned, as animal studies, as well as a human trial, showed a decrease in serum cholesterol and no effects or a decrease in plasma triglycerides. To obtain more insight into the effects of PFASs on these processes, the present study investigated the effects of PFOA, PFOS, and perfluorononanoic acid (PFNA) on intracellular triglyceride and cholesterol levels in human HepaRG liver cells. DNA microarray analyses were performed to provide insight into underlying mechanisms. All PFASs induced an increase in cellular triglyceride levels, but had no effect on cholesterol levels. Gene set enrichment analysis (GSEA) of the microarray data indicated that gene sets related to cholesterol biosynthesis were repressed by PFOA, PFOS, and PFNA. Other gene sets commonly affected by all PFAS were related to PERK/ATF4 signaling (induced), tRNA amino-acylation (induced), amino acid transport (induced), and glycolysis/gluconeogenesis (repressed). Moreover, numerous target genes of peroxisome proliferator-activated receptor α (PPARα) were found to be upregulated. Altogether, the present study shows that PFOA, PFOS, and PFNA increase triglyceride levels and inhibit cholesterogenic gene expression in HepaRG cells. In addition, the present study indicates that PFASs induce endoplasmic reticulum stress, which may be an important mechanism underlying some of the toxic effects of these chemicals.

Toward on-site food authentication using nanopore sequencing.

•MinION DNA metabarcoding is a promising tool for species identification in food.•MinION and Illumina MiSeq sequencing platforms perform equally accurate.•Species identification with MinION sequencing requires dedicated bioinformatics.

Omics analyses of potato plant materials using an improved one-class classification tool to identify aberrant compositional profiles in risk assessment procedures.

The objective of this study was to quantitatively assess potato omics profiles of new varieties for meaningful differences from analogous profiles of commercial varieties through the SIMCA one-class classification model. Analytical profiles of nine commercial potato varieties, eleven experimental potato varieties, one GM potato variety that had acquired Phytophtora resistance based on a single insert with potato-derived DNA sequences, and its non-GM commercial counterpart were generated. The ten conventional varieties were used to construct the one-class model. Omics profiles from experimental non-GM and GM varieties were assessed using the one-class SIMCA models. No potential unintended effects were identified in the case of the GM variety. The model showed that varieties that were genetically more distant from the commercial varieties were recognized as aberrant, highlighting its potential in determining whether additional evaluation is required for the risk assessment of materials produced from any breeding technique, including genetic modification.

Use of omics analytical methods in the study of genetically modified maize varieties tested in 90 days feeding trials.

Genetically modified (GM) maize and their non-modified counterparts were compared using MON810 varieties, the only GMO event cultivated in Europe. The differences in grain samples were analysed by omics profiles, including transcriptomics, proteomics and metabolomics. Other cultivated maize varieties were analysed as a reference for the variability that will exist between cultivated varieties. The observed differences between modified and non-modified maize varieties do not exceed typical differences between non-modified varieties. The use of these advanced analytical approaches to analyse novel plant materials as compared to the results from animal feeding trials with whole foods is assessed. No indications were observed for changes in the GM varieties that warrant further investigations. Furthermore, it was shown that such indications will be obtained if maize samples of inferior quality are analysed similarly. Omics data provide detailed analytical information of the plant material, which facilitates a risk assessment procedure of new (GM) plant varieties.

Comparative genomics of plant pathogenic Botrytis species with distinct host specificity.

BACKGROUND: Fungi of the genus Botrytis (presently containing ~ 35 species) are able to infect more than 1400 different plant species and cause losses in a wide range of crops of economic importance. The best studied species is B. cinerea, which has a broad host range and is one of the best studied necrotrophic plant pathogenic fungi. Most other Botrytis spp. have a narrow host range and have been studied in less detail. To characterize genomic variation among different representatives of Botrytis spp., we sequenced and annotated the draft genomes of nine Botrytis species: B. calthae, B. convoluta, B. elliptica, B. galanthina, B. hyacinthi, B. narcissicola, B. paeoniae, B. porri and B. tulipae. RESULTS: Bioinformatics and comparative genomics tools were applied to determine a core of 7668 shared protein families in all Botrytis species, which grouped them in two distinct phylogenetic clades. The secretome of all nine Botrytis spp. was similar in number (ranging from 716 to 784 predicted proteins). A detailed analysis of the molecular functions of the secretome revealed that shared activities were highly similar. Orthologs to effectors functionally studied in B. cinerea were also present in the other Botrytis species. A complex pattern of presence/absence of secondary metabolite biosynthetic key enzymes was observed. CONCLUSIONS: Comparative genomics of Botrytis show that overall, species share the main signatures and protein families in the secreted proteins, and of known effectors. Our study provides leads to study host range determinants in the genus Botrytis and provides a stepping stone to elucidate the roles of effector candidates in the infection process of these species.

Author Correction: ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis.

The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

Big data in food safety: An overview.

Technology is now being developed that is able to handle vast amounts of structured and unstructured data from diverse sources and origins. These technologies are often referred to as big data, and open new areas of research and applications that will have an increasing impact in all sectors of our society. In this paper we assessed to which extent big data is being applied in the food safety domain and identified several promising trends. In several parts of the world, governments stimulate the publication on internet of all data generated in public funded research projects. This policy opens new opportunities for stakeholders dealing with food safety to address issues which were not possible before. Application of mobile phones as detection devices for food safety and the use of social media as early warning of food safety problems are a few examples of the new developments that are possible due to big data.

Advances in DNA metabarcoding for food and wildlife forensic species identification.

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.

Authentication of Closely Related Fish and Derived Fish Products Using Tandem Mass Spectrometry and Spectral Library Matching.

Proteomics methodology has seen increased application in food authentication, including tandem mass spectrometry of targeted species-specific peptides in raw, processed, or mixed food products. We have previously described an alternative principle that uses untargeted data acquisition and spectral library matching, essentially spectral counting, to compare and identify samples without the need for genomic sequence information in food species populations. Here, we present an interlaboratory comparison demonstrating how a method based on this principle performs in a realistic context. We also increasingly challenge the method by using data from different types of mass spectrometers, by trying to distinguish closely related and commercially important flatfish, and by analyzing heavily contaminated samples. The method was found to be robust in different laboratories, and 94-97% of the analyzed samples were correctly identified, including all processed and contaminated samples.

Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L.

Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP.

The Leguminosae has emerged as a model for studying angiosperm plastome evolution because of its striking diversity of structural rearrangements and sequence variation. However, most of what is known about legume plastomes comes from few genera representing a subset of lineages in subfamily Papilionoideae. We investigate plastome evolution in subfamily Mimosoideae based on two newly sequenced plastomes (Inga and Leucaena) and two recently published plastomes (Acacia and Prosopis), and discuss the results in the context of other legume and rosid plastid genomes. Mimosoid plastomes have a typical angiosperm gene content and general organization as well as a generally slow rate of protein coding gene evolution, but they are the largest known among legumes. The increased length results from tandem repeat expansions and an unusual 13 kb IR-SSC boundary shift in Acacia and Inga. Mimosoid plastomes harbor additional interesting features, including loss of clpP intron1 in Inga, accelerated rates of evolution in clpP for Acacia and Inga, and dN/dS ratios consistent with neutral and positive selection for several genes. These new plastomes and results provide important resources for legume comparative genomics, plant breeding, and plastid genetic engineering, while shedding further light on the complexity of plastome evolution in legumes and angiosperms.

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing.

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Repeated loss of an anciently horizontally transferred gene cluster in Botrytis.

At least five of the six genes of the bikaverin secondary metabolic gene cluster were shown to have undergone horizontal transfer (HGT) from a Fusarium donor to the Botrytis lineage. Of these five, two enzyme-encoding genes are found as pseudogenes in B. cinerea whereas two regulatory genes and the transporter remain intact. To reconstruct the evolutionary events leading to decay of this gene cluster and infer a more precise timing of its transfer, we examined the genomes of nine additional broadly sampled Botrytis species. We found evidence that a Botrytis ancestor acquired the entire gene cluster through an ancient HGT that occurred before the diversification of the genus. During the subsequent evolution and diversification of the genus, four of the 10 genomes appear to have lost the gene cluster, while in the other six the cluster is in various stages of degeneration. Across the Botrytis genomes, the modes of gene decay in the cluster differed between enzyme-encoding genes, which had higher rates of transition to or retention of pseudogenes and were universally inactivated, and regulatory genes (particularly the non-pathway-specific regulator bik4), which more frequently appeared intact. Consistent with these results, the regulatory genes bik4 and bik5 showed stronger evidence of transcriptional expression than other bikaverin genes under multiple conditions in B. cinerea. These results could be explained by pleiotropy in the bikaverin regulatory genes either through rewiring or their interaction with more central pathways or by constraints on the order of gene loss driven by the intrinsic toxicity of the pathway. Our finding that most of the bikaverin pathway genes have been lost or pseudogenized in these Botrytis genomes suggests that the incidence of HGT of gene cluster-encoded metabolic pathways might be higher than what is possible to be inferred from isolated genome analyses.

Genome update of Botrytis cinerea strains B05.10 and T4.

We report here an update of the Botrytis cinerea strains B05.10 and T4 genomes, as well as an automated preliminary gene structure annotation. High-coverage de novo assemblies and reference-based alignments led to a correction of wrong base calls, elimination of sequence gaps, and improved joining of contigs. The new assemblies have substantially lower numbers of scaffolds and a concomitant increase in the N(50).The list of protein-coding genes was generated using the evidence-driven gene predictor Augustus, with expressed sequence tag evidence and RNA-Seq data as input.

How to open the treasure chest? Optimising DNA extraction from herbarium specimens.

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200-300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.