Molecular changes in agroinfiltrated leaves of Nicotiana benthamiana expressing suppressor of silencing P19 and coronavirus‐like particles

Summary The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana . Relying on bacterial vector Agrobacterium tumefaciens , this process is favoured by co‐expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus‐like particles (CoVLPs) occurs. We previously characterized the effects of influenza virus hemagglutinin forming VLPs through similar processes. However, leaf samples were only collected after 6 days of expression, and it is unknown whether influenza VLPs (HA‐VLPs) and CoVLPs induce similar responses. Here, time course sampling was used to profile responses of N. benthamiana leaf cells expressing P19 only, or P19 and the S protein. The latter triggered early but transient activation of the unfolded protein response and waves of transcription factor genes involved in immunity. Accordingly, defence genes were induced with different expression kinetics, including those promoting lignification, terpene biosynthesis, and oxidative stress. Cross‐talk between stress hormone pathways also occurred, including repression of jasmonic acid biosynthesis genes after agroinfiltration, and dampening of salicylic acid responses upon S protein accumulation. Overall, HA‐VLP‐ and CoVLP‐induced responses broadly overlapped, suggesting nanoparticle production to have the most effects on plant immunity, regardless of the virus surface proteins expressed. Taking advantage of RNAseq inferences, we finally show the co‐expression of Kunitz trypsin inhibitors to reduce CoVLP‐induced defence and leaf symptoms, with no adverse effect on plant productivity.

Expression of a constitutively active nitrate reductase increases SARS‐CoV‐2 Spike protein production in Nicotiana benthamiana leaves that otherwise show traits of senescence

Summary The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the Spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana , a process promoted by the co‐expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus‐like particles (CoVLPs) occurs. We recently used RNAseq and time course sampling to characterize molecular responses of N. benthamiana leaf cells expressing P19 only or P19 in combination with recombinant S protein. This revealed expression of the viral proteins to deeply affect the physiological status of plant cells, including through the activation of immune responses. Here, transcriptomics shows that the production of CoVLPs also induces leaf senescence, as revealed by the up‐regulation of senescence‐associated genes, activation of senescence‐related proteases and down‐regulation of genes involved in basic metabolic functions like photosynthesis or nitrogen uptake and assimilation. CoVLP production also up‐regulates asparagine synthetase genes and leads to the consequent accumulation of asparagine, a nitrogen‐rich amino acid known to facilitate reallocation of nitrogen resources from senescent to young growing organs. Hypothesizing these combined host responses to restrain foreign protein accumulation, an attempt was made to support nitrogen reduction in CoVLP‐producing leaves by co‐expressing a constitutively active, light‐insensitive form of nitrate reductase. We show this strategy to increase S protein accumulation in leaf tissues, thereby suggesting that boosting nitrogen metabolism in agroinfiltrated leaves may improve recombinant protein yields in N. benthamiana .

The Nicotiana benthamiana Biofactory

Early osteopathic treatment delivered to patients with an acute lateral ankle sprain improves recovery: an investigative study

Initiating transformative medical practices to address environmental challenges – proceedings of “Santé en 2050”, the French national conference on healthcare adaptation

“Santé en 2050” is the first medical conference in France dedicated to the adaptation of healthcare systems to environmental constraints. Organized in 2024 and 2025 by healthcare professionals within The Shifters, a non-profit association committed to raising public awareness of the dual carbon challenge, the conference provided both a scientific platform for research on mitigation and adaptation in health and a forum to enhance professional competencies on these cross-cutting issues. Over its two successful editions, the event mapped the emerging disease landscape driven by environmental exposures and climate change, affecting both communicable and non-communicable diseases due to shifting temperatures, precipitation patterns, mass population movements, unhealthy lifestyles, and increasing pollution. Climate change also poses indirect threats to health by straining infrastructure, disrupting supply chains, and triggering crises that practitioners must address with ever-scarcer resources. The conference underscored the urgent need for a systemic overhaul of healthcare to build resilient, sustainable care pathways. It provided attendees with practical tools to integrate ecodesigned care, integrative medical approaches, and non-pharmacological interventions into their daily practice. The conference further explored pressing ethical and philosophical dilemmas, examining how technology, research priorities, and innovation must evolve in response to environmental constraints and the broader responsibility of medicine at a population level. Here we summarize the topics discussed during the two editions of the conference. By equipping healthcare workers with skills in epidemiology, disease prevention, and continuity of care amid accelerating environmental change, “Santé en 2050” aims to prepare the medical community for the challenges of tomorrow.

Safety of carbon dioxide extract from Cannabis sativa L. as a novel food pursuant to Regulation ( EU ) 2015/2283

Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on a carbon dioxide (CO 2 ) extract from industrial hemp varieties of the plant Cannabis sativa L. as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is proposed to be used in food supplements at maximum use levels ranging from 1.12 mg/day for children above 3 years of age to 3.60 mg/day for adults, excluding pregnant and lactating women. The NF contains cannabidiol (CBD) as well as other cannabinoids. The Panel notes that a substantial proportion of the NF remains uncharacterised. Stability and toxicological tests submitted by the applicant were performed with a material different from the NF under assessment, as it was produced using a different production process (i.e. isopropanol (IPA)‐based extraction instead of CO 2 supercritical extraction), potentially resulting in different compositional data. Therefore, the absence of adequate stability and of toxicological studies performed with a representative test item prevents the Panel from drawing conclusions on the safety assessment of the NF. Additionally, no data were provided by the applicant to address the safety of the NF in the general population above 3 years of age. Therefore, the safety of the NF under the proposed conditions of use cannot be established.

Safety of 3′‐sialyllactose (3′‐ SL ) sodium salt produced with a derivative strain ( Escherichia coli K‐12 MG1655 INB ‐3SL_01) of E. coli K‐12 MG1655 ( ATCC 700926) as a novel food pursuant to Regulation ( EU ) 2015/2283

Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens ( NDA ) was asked to deliver an opinion on 3′‐sialyllactose (3′‐ SL ) sodium salt as a novel food ( NF ) pursuant to Regulation ( EU ) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharide ( HiMO ) 3′‐ SL (sodium salt), but it also contains d ‐lactose, sialic acid, 3′‐sialyllactulose and a small fraction of other related saccharides. The NF is produced by fermentation with a genetically modified strain ( Escherichia coli K‐12 MG1655 INB ‐3SL_01) of E. coli K‐12 MG1655 ( ATCC 700926). 3′‐ SL (sodium salt) produced by fermentation with genetically modified strains of E. coli K‐12 DH1 , E. coli BL21 ( DE3 ) or E. coli W ( ATCC 9637) is already authorised and included in the EU list of NFs . This application refers to a change in the production process and specifications, while the target population, conditions of use and consequently the anticipated intake remain unchanged. The information provided on the identity, production process, composition and specifications and the toxicological data of the NF do not raise safety concerns. The intake of other carbohydrate‐type compounds structurally related to 3′‐ SL is also considered of no safety concern. In line with other HiMOs , the safety assessment of this NF is mainly based on the comparison between the intake by breastfed infants and the estimated intake of the NF . Given that the NF would be consumed to the same extent as the already authorised 3′‐ SL (sodium salt), the Panel considers that the consumption of the NF at the proposed uses and use levels does not raise safety concerns. The Panel concludes that the NF is safe under the proposed conditions of use

Safety of 6′‐sialyllactose (6′‐ SL ) sodium salt produced with a derivative strain ( Escherichia coli K‐12 MG1655 INB ‐6SL_02) of E. coli K‐12 MG1655 ( ATCC 700926) as a novel food pursuant to Regulation ( EU ) 2015/2283

Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on 6′‐sialyllactose (6’‐SL) sodium salt as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharide (HiMO) 6’‐SL (sodium salt), but it also contains d ‐lactose, sialic acid, 6′‐sialyllactulose and a small fraction of other related saccharides. The NF is produced by fermentation with a genetically modified strain ( Escherichia coli K‐12 MG1655 INB‐6SL_02) of E. coli K‐12 MG1655 (ATCC 700926). 6’‐SL (sodium salt) produced by fermentation with genetically modified strains of E. coli K‐12 DH1, E. coli BL21 or E. coli W is already authorised and included in the EU list of NFs. This application refers to a change in the production process and specifications, while the target population, conditions of use and consequently the anticipated intake remain unchanged. The information provided on the identity, production process, composition and specifications and the toxicological data of the NF do not raise safety concerns. The intake of other carbohydrate‐type compounds structurally related to 6’‐SL is also considered of no safety concern. In line with other HiMOs, the safety assessment of this NF is mainly based on the comparison between the intake of breastfed infants and the estimated intake of the NF. Given that the NF would be consumed to the same extent as the already authorised 6’‐SL (sodium salt), the Panel considers that the consumption of the NF at the proposed uses and use levels does not raise safety concerns. The Panel concludes that the NF is safe under the proposed conditions of use.

Scientific Opinion on the tolerable upper intake level for supplemental docosahexaenoic acid

Abstract Following a request from the European Commission, the Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the revision of the safe level of intake for supplemental docosahexaenoic acid (DHA). Systematic reviews of the literature were conducted to identify human intervention studies administering supplemental DHA alone from a source with an eicosapentaenoic acid (EPA)/DHA ratio < 0.3 for at least 8 weeks, without restrictions on population or outcome. Hazard identification focused on bleeding complications (including bleeding time, platelet function and blood clotting parameters), glucose homeostasis, blood lipid profile, markers of lipid peroxidation, immune function, pregnancy endpoints and safety, tolerability and adverse events. The risk of spontaneous bleeding was selected as the critical effect on which to base the UL/safe level of intake for supplemental DHA alone. In the absence of adequate data to characterise a dose–response relationship and identify a reference point, no UL for supplemental DHA alone can be established for any population group. Therefore, the Panel derived a safe level of intake, which differs from a UL in that it is based on intakes up to which no adverse effects have been observed. Based on the available evidence, the Panel retains the safe level of intake of 1 g/day for supplemental DHA alone established in 2012 for all population groups (i.e. infants, children, adolescents and adults, including pregnant and lactating women). This safe level of intake applies to DHA added to foods or consumed as food supplements in any chemical form (e.g. triacylglycerols, ethyl esters, phospholipids) from sources (e.g. fish oil concentrates, algal oils, krill oils) containing DHA alone or mostly DHA (i.e., EPA/DHA ratio < 0.3).

Update of the statement on safety of cannabidiol as a novel food

Abstract During the assessment of cannabidiol (CBD) as a novel food, in 2022 the NDA Panel identified significant data gaps. Concerns focused on potential adverse effects on the liver, gastrointestinal tract, endocrine, nervous and reproductive systems. Literature searches covering animal and human studies from the previous Statement until June 2024 confirmed the persistence of these gaps, as many of the new studies suffer from methodological limitations, including non‐standardised protocols, short durations and concomitant treatment with medicine. Pharmacokinetic studies confirmed that CBD's bioavailability is variable, influenced by delivery matrix and food intake. Its ability to cross the placenta and accumulate systemically raises further safety concerns. Animal studies revealed consistent liver toxicity, with liver weight and histopathological changes emerging as sensitive endpoints. Human trials indicated hepatotoxic potential, particularly when CBD is used in combination with other medications. Gastrointestinal effects were reported at higher doses, while neurological and psychiatric safety data remain insufficient. Animal studies on reproductive toxicity reinforced the concern regarding this endpoint. Neurodevelopmental effects following prenatal exposure were observed, suggesting long‐lasting, sex‐specific outcomes. Endocrine disruptions were noted, including altered thyroid hormone levels and adrenal histopathology. No studies addressed immunotoxicity, though CBD's interaction with immune pathways warrants caution. The Panel performed benchmark dose modelling based on GLP‐compliant subchronic studies to identify a toxicological reference point. By applying an uncertainty factor of 400, a provisional safe dose of 0.0275 mg/kg bw per day (approximately 2 mg/day for a 70 kg adult) was derived. This provisional safe dose applies solely to food supplement formulations with CBD purity ≥ 98%, without nanoparticles, for which the production process is considered safe and genotoxicity is ruled out. The Panel concludes that, based on all available data, the safety of CBD for individuals under 25 years of age, pregnant or lactating women, and those on concurrent medications, cannot be established.