Biosafety of GM Crop Plants Expressing dsRNA:Data Requirements and EU Regulatory Considerations

The use of RNA interference (RNAi) enables the silencing of target genes in plants or plant-dwelling organisms, through the production of double stranded RNA (dsRNA) resulting in altered plant characteristics. Expression of properly synthesized dsRNAs in plants can lead to improved crop quality characteristics or exploit new mechanisms with activity against plant pests and pathogens. Genetically modified (GM) crops exhibiting resistance to viruses or insectsviaexpression of dsRNA have received authorization for cultivation outside Europe. Some products derived from RNAi plants have received a favourable opinion from the European Food Safety Authority (EFSA) for import and processing in the European Union (EU). The authorization process in the EU requires applicants to produce a risk assessment considering food/feed and environmental safety aspects of living organisms or their derived food and feed products. The present paper discusses the main aspects of the safety assessment (comparative assessment, molecular characterization, toxicological assessment, nutritional assessment, gene transfer, interaction with target and non-target organisms) for GM plants expressing dsRNA, according to the guidelines of EFSA. Food/feed safety assessment of products from RNAi plants is expected to be simplified, in the light of the consideration that no novel proteins are produced. Therefore, some of the data requirements for risk assessment do not apply to these cases, and the comparative compositional analysis becomes the main source of evidence for food/feed safety of RNAi plants. During environmental risk assessment, the analysis of dsRNA expression levels of the GM trait, and the data concerning the observable effects on non-target organisms (NTO) will provide the necessary evidence for ensuring safety of species exposed to RNAi plants. Bioinformatics may provide support to risk assessment by selecting target gene sequences with low similarity to the genome of NTOs possibly exposed to dsRNA. The analysis of these topics in risk assessment indicates that the science-based regulatory process in Europe is considered to be applicable to GM RNAi plants, therefore the evaluation of their safety can be effectively conducted without further modifications. Outcomes from the present paper offer suggestions for consideration in future updates of the EFSA Guidance documents on risk assessment of GM organisms

Search for black holes and other new phenomena in high-multiplicity final states in proton-proton collisions at root s=13 TeV

Peer reviewe

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins

Ontologies Enriched among the 83 Genes That Are Essential in Yeast Expressing OspF

<p>A black cell indicates membership of its column's gene in its row's ontology. Two independent types of hierarchy are represented in the matrix. Columns and rows have been hierarchically clustered and the dendrogram on the left shows the results of row clustering (column dendrogram not shown). Gene ontologies are hierarchical classifications, and the color bands indicate hierarchically related groups of process ontologies (in bold type). Ontologies in italics are molecular functions; the remaining five are components.</p

OspF Inhibits ERK and p38 Phosphorylation

<div><p>(A) Immunoblots of extracts of HeLa cells infected with wild-type, <i>ΔospF,</i> or <i>ΔospF/</i>pOspF <i>Shigella</i> for 1 h.</p><p>(B) pOspF indicates the OspF complementing plasmid. Cell lysates were probed with the designated antibodies. Immunoblots of extracts of uninfected HeLa cells or HeLa infected with wild-type or <i>ΔospF Shigella</i> for 1 h and then exposed to 50 ng/ml EGF or 0.4 M sorbitol to activate ERK and p38 signaling, respectively.</p><p>(C) Immunoblots of extracts of HeLa cells infected with wild-type <i>Shigella</i> (MOI 10:1), <i>ΔospF Shigella</i> (MOI 10:1), or mix of wild-type <i>Shigella</i> (MOI 5:1) plus <i>ΔospF Shigella</i> (MOI 5:1) for 1 h. In all cases, each experiment was conducted at least in triplicate with similar results.</p></div

OspF Is Associated with Attenuation of the Host Innate Immune Response

<p>The images shown are of hematoxylin- and eosin-stained sections of lungs of mice 24 h after infection with wild-type or <i>ΔospF Shigella</i> or injection with equivalent volume of phosphate buffered saline.</p

OspF Inhibits the Yeast CWI Pathway by Inhibition of MAPK Phosphorylation

<div><p>(A) Summary of the activity of an RLM1-regulated β-galactosidase reporter in response to heat shock in the presence or absence of OspF.</p><p>(B) Outline of the CWI pathway.</p><p>(C) Yeast containing empty vector or a plasmid that conditionally expresses OspF were subjected to the designated stress 2 h after the induction of expression of OspF (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030021#s4" target="_blank">Materials and Methods</a>). Representative immunoblots used to assay for activation of each of four MAPK signaling pathways: SLT2 (CWI pathway), FUS3 (mating pathway), KSS1 (invasive growth pathway), and HOG1 (high-osmolarity glycerol pathway) are shown. The circled P denotes phosphorylated versions of the proteins. The blots were probed with the anti-PSTAIRE antibody that recognizes CDC28 and PHO85 as a loading control. Each experiment was conducted at least in triplicate with similar results.</p></div