Clustering and classification of reference documents from large-scale literature searches: Support to the SAM explanatory note "New Techniques in Agricultural Biotechnology"

Searches of the existing scientific literature are the cornerstone of scientific research and reporting. With the constant growth of the rate at which scientific studies and reviews are being published, the information produced by these searches can be delicate to manage. Narrowing the search increases the risk of overlooking important documents, while broadening it can produce too many documents to be reasonably processed. This report describes a set of strategies designed to process large sets of scientific references (such as those obtained by broad literature searches) and assist in the identification of documents relevant for specific purposes. These strategies take advantage of metadata associated to each document in SCOPUS, the database of peer-reviewed literature maintained by Elsevier and accessible through an Application Programming Interface (API). These strategies were developed and applied in support to the European Commission's Scientific Advice Mechanism (SAM) in managing the results of literature searches in the context of the exploratory note "New Techniques in Agricultural Biotechnology".JRC.F.7-Knowledge for Health and Consumer Safet

Literature and Bioinformatics Analyses of Wheat-specific Detection Methods

In view of the recent necessity to perform testing for the detection of genetically modified common wheat (Triticum aestivum), the need arises for a taxon-specific method for this organism. However, no such method has yet been officially validated. Multiple species of wheat exist on the market, such as common wheat, durum wheat, emmer wheat, etc. These plants have complex genomes, composed of different combinations (from diploid to hexaploid) of common sets of chromosomes. The specificity of a method then depends on which set of chromosome the targeted region is located, which increases the complexity of identifying methods specific to Triticum aestivum. Often, such methods were developed for the specific regulatory need of differentiating durum and common wheat (for example, in alimentary pasta labeling), with minimal concerns for non-specific detection of other plants. This document summarises the review performed by the EU-RL GMFF, complemented with in-house bioinformatics analyses, in order to identify and characterise Triticum aestivum-specific detection methods that have been described in the scientific literature. Methods with apparent specificity (based on results shown and bioinformatics analyses) and promising performance (based on results shown) are highlighted and their primers and probe sequences reported. Those methods are the 'SS II-D' and ' SS II ex7' methods described in Matsuoka et al. (2012) and the 'wx012' method described in Iida et al. (2005), and they represent good candidates to uniquely identify common wheat in complex food samples.JRC.I.3-Molecular Biology and Genomic

Systematic identification of stem-loop containing sequence families in bacterial genomes

<p>Abstract</p> <p>Background</p> <p>Analysis of non-coding sequences in several bacterial genomes brought to the identification of families of repeated sequences, able to fold as secondary structures. These sequences have often been claimed to be transcribed and fulfill a functional role. A previous systematic analysis of a representative set of 40 bacterial genomes produced a large collection of sequences, potentially able to fold as stem-loop structures (SLS). Computational analysis of these sequences was carried out by searching for families of repetitive nucleic acid elements sharing a common secondary structure.</p> <p>Results</p> <p>The initial clustering procedure identified clusters of similar sequences in 29 genomes, corresponding to about 1% of the whole population. Sequences selected in this way have a substantially higher aptitude to fold into a stable secondary structure than the initial set. Removal of redundancies and regrouping of the selected sequences resulted in a final set of 92 families, defined by HMM analysis. 25 of them include all well-known SLS containing repeats and others reported in literature, but not analyzed in detail. The remaining 67 families have not been previously described. Two thirds of the families share a common predicted secondary structure and are located within intergenic regions.</p> <p>Conclusion</p> <p>Systematic analysis of 40 bacterial genomes revealed a large number of repeated sequence families, including known and novel ones. Their predicted structure and genomic location suggest that, even in compact bacterial genomes, a relatively large fraction of the genome consists of non-protein-coding sequences, possibly functioning at the RNA level.</p

Stem-loop structures in prokaryotic genomes

BACKGROUND: Prediction of secondary structures in the expressed sequences of bacterial genomes allows to investigate spontaneous folding of the corresponding RNA. This is particularly relevant in untranslated mRNA regions, where base pairing is less affected by interactions with the translation machinery. Relatively large stem-loops significantly contribute to the formation of more complex secondary structures, often important for the activity of sequence elements controlling gene expression. RESULTS: Systematic analysis of the distribution of stem-loop structures (SLSs) in 40 wholly-sequenced bacterial genomes is presented. SLSs were searched as stems measuring at least 12 bp, bordering loops 5 to 100 nt in length. G-U pairing in the stems was allowed. SLSs found in natural genomes are constantly more numerous and stable than those expected to randomly form in sequences of comparable size and composition. The large majority of SLSs fall within protein-coding regions but enrichment of specific, non random, SLS sub-populations of higher stability was observed within the intergenic regions of the chromosomes of several species. In low-GC firmicutes, most higher stability intergenic SLSs resemble canonical rho-independent transcriptional terminators, but very frequently feature at the 5'-end an additional A-rich stretch complementary to the 3' uridines. In all species, a clearly biased SLS distribution was observed within the intergenic space, with most concentrating at the 3'-end side of flanking CDSs. Some intergenic SLS regions are members of novel repeated sequence families. CONCLUSION: In depth analysis of SLS features and distribution in 40 different bacterial genomes showed the presence of non random populations of such structures in all species. Many of these structures are plausibly transcribed, and might be involved in the control of transcription termination, or might serve as RNA elements which can enhance either the stability or the turnover of cotranscribed mRNAs. Three previously undescribed families of repeated sequences were found in Yersiniae, Bordetellae and Enterococci

Report on the Verification of the Performance of a Method for the Detection of Event MON71800 in Wheat Using Real-Time PCR

Following the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS) announcement that test results confirmed the finding of unauthorised GM glyphosate-resistant wheat "volunteer" plants harbouring the event MON71800 on a farm in Oregon, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) was requested to provide as soon as possible a method to test wheat consignments for the presence of this genetically modified organism (GMO) to the National Reference Laboratories (NRLs) for GMOs of the EU Member States. In response, the EU-RL put together a testing strategy, based on readily available screening tests which was published here (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm). Upon request, Monsanto provided in May 2013 the EU-RL with the procedure “Roundup Ready® Wheat MON71800 Event Specific Endpoint TaqMan® PCR with acc Internal Control for Seed Pools of 1:15” that had previously been made available to, and was used by USDA. The EU-RL GMFF tested this protocol on positive control samples consisting of MON71800 crude lysate, also provided by Monsanto. Our results can be summarised as follow: The method is apparently event-specific. Our specificity-tests did not show cross-reactivity on genomic DNA from a wide selection of similar GMO. The sensitivity of the method was found to be in agreement with previous findings of USDA, i.e. the relative limit of detection lies at 0.5% in a background of 301 ng of total wheat genomic DNA. The absolute limit of detection (LODabs) was determined between 5 and 10 copies of MON71800 target. The latter was not indicated by the USDA. For seed/grains the application of a sub-sampling strategy could allow detection below 0.5% but would require significant additional efforts, including the analysis of numerous sub-samples. Our tests also indicated that the duplex PCR system at the tested stage of optimisation is characterised by poor efficiency at increasing background DNA concentration in reaction. Based on the scientific evidence described in the present report, the EU-RL suggest that its testing strategy (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm), making use of validated element and construct-specific methods and found to be more sensitive, is used to test for presence of MON71800 GM-wheat. The verified event specific method of Monsanto could be used to confirm positive findings at GM-target concentration equal or above 0.5% or it could be used for detection of GM-event MON71800 below 0.5% but it would require a costly sub-sampling strategy, which, in addition, is only possible in seeds/grains.JRC.I.3-Molecular Biology and Genomic

Mechanisms leading to gut dysbiosis in COVID-19: current evidence and uncertainties based on adverse outcome pathways

Alteration in gut microbiota has been associated with COVID-19. However, the underlying mechanisms remain poorly understood. Here, we outlined three potential interconnected mechanistic pathways leading to gut dysbiosis as an adverse outcome following SARS-CoV-2 presence in the gastrointestinal tract. Evidence from the literature and current uncertainties are reported for each step of the different pathways. One pathway investigates evidence that intestinal infection by SARS-CoV-2 inducing intestinal inflammation alters the gut microbiota. Another pathway links the binding of viral S protein to angiotensin-converting enzyme 2 (ACE2) to the dysregulation of this receptor, essential in intestinal homeostasis—notably for amino acid metabolism—leading to gut dysbiosis. Additionally, SARS-CoV-2 could induce gut dysbiosis by infecting intestinal bacteria. Assessing current evidence within the Adverse Outcome Pathway framework justifies confidence in the proposed mechanisms to support disease management and permits the identification of inconsistencies and knowledge gaps to orient further research.info:eu-repo/semantics/publishedVersio

The first report on detecting SARS-CoV-2 inside human fecal-oral bacteria: A case series on asymptomatic family members and a child with COVID-19

Many studies report the importance of using feces as source sample for detecting SARS-CoV-2 in patients with COVID-19 symptoms but who are negative to oropharyngeal/ nasopharyngeal tests. Here, we report the case of an asymptomatic child whose family members had negative results with the rapid antigen nasopharyngeal swab tests. The 21-month-old child presented with fever, diarrhea, bilateral conjunctivitis, and conspicuous lacrimation. In this study, analysis for the presence of SARS-CoV-2 in fecal samples by using Luminex technology allowed accurate detection of the presence of the viral RNA in the feces of the child and of all her relatives, which thus resulted to be positive but asymptomatic. It is the first time that SARS-CoV-2- is observed inside human fecal-oral bacteria and outside a matrix resembling extracellular bacterial lysates, in agreement with a bacteriophage mechanism with the images obtained by transmission electron microscopy (TEM), post-embedding immunogold, and by fluorescence microscope. In addition to the typical observations of respiratory symptoms, accurate evaluation of clinical gastrointestinal and neurological symptoms, combined with efficient highly sensitive molecular testing on feces, represent an efficient approach for detecting SARS-CoV-2, and for providing the correct therapy in challenging COVID-19 cases, like the one here reported

The first report on detecting SARS-CoV-2 inside bacteria of the human gut microbiome: A case series on asymptomatic family members and a child with COVID-19

Many studies report the importance of using feces as source sample for detecting SARS-CoV-2 in patients with COVID-19 symptoms but who are negative to oropharyngeal/ nasopharyngeal tests. Here, we report the case of an asymptomatic child whose family members had negative results with the rapid antigen nasopharyngeal swab tests. The 21-month-old child presented with fever, diarrhea, bilateral conjunctivitis, and conspicuous lacrimation. In this study, analysis for the presence of SARS-CoV-2 in fecal samples by using Luminex technology allowed accurate detection of the presence of the viral RNA in the feces of the child and of all her relatives, which thus resulted to be positive but asymptomatic. It is the first time that SARS-CoV-2- is observed inside bacteria of the human gut microbiome and outside a matrix resembling extracellular bacterial lysates, in agreement with a bacteriophage mechanism with the images obtained by transmission electron microscopy (TEM), post-embedding immunogold, and by fluorescence microscope. In addition to the typical observations of respiratory symptoms, accurate evaluation of clinical gastrointestinal and neurological symptoms, combined with efficient highly sensitive molecular testing on feces, represent an efficient approach for detecting SARS-CoV-2, and for providing the correct therapy in challenging COVID-19 cases, like the one here reported

Report on the Verification of the Performance of a Testing Strategy for the Detection of Wheat MON71800 Event Using Real-Time PCR

In response to a request of DG SANCO to provide National Reference Laboratories (NRLs) as soon as possible with a method to test soft white wheat consignments for the presence of unauthorised GM glyphosate-resistant wheat harbouring the event MON71800, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) developed, in collaboration with the European Network of GMO Laboratories (ENGL), a testing strategy intended to be immediately implementable by EU NRLs. The testing strategy is based on a combination of three validated screening methods that allow excluding (detectable) presence of Monsanto’s GM glyphosate-resistant wheat (MON71800) in wheat grain or food/feed products and confirming its presence whenever other GMOs can be excluded. The present report describes the results of the tests carried out by the EU-RL GMFF to verify the testing strategy proposed; the tests were conducted using the positive control sample represented by a crude DNA lysate of MON71800 provided by Monsanto and genomic DNA samples of genetically modified organisms harbouring the CTP2-CP4epsps element for which a validated event-specific method is available. The sensitivity of the three methods was assessed by verifying the relative limit of detection (LODrel) on MON71800 wheat DNA. The LODrel is approximately 0.03% for the P-35S and for T-nos methods and 0.06% for the CTP2-CP4epsps method in 300 nanograms of wheat genomic DNA. Further experimental evidence confirmed that the three methods react against genomic DNA extracted from GM events containing the CTP2-CP4epsps element for which a validated event-specific method is available. The experimental verification hereby reported confirmed the validity of the EU-RL GMFF guidance on testing for GM glyphosate-resistant wheat (MON71800) in wheat grain or in food/feed products containing wheat flour originating or consigned from the US, provided that DNA of acceptable quality can be obtained.JRC.I.3-Molecular Biology and Genomic

Systematic analysis of human kinase genes: a large number of genes and alternative splicing events result in functional and structural diversity

BACKGROUND: Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosys. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. RESULTS: A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was perfomed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. CONCLUSION: The predicted human kinome was extended by identifiying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set