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Abstract Objective The purpose of this study was to evaluate the in vitro antibacterial effects of a p‐Cymene‐based bis(pyrazolyl)methane derivative (SC‐19) to advance in developing alternative therapeutic compounds to fight against bacterial isolates from patients with otitis externa (OE). Methods Eighteen swab specimens were collected from patients aged over 18 years diagnosed with OE within at least 7 days of symptom onset, contaminated by only one bacterium type: Pseudomonas aeruginosa (n = 5); Staphylococcus aureus (n = 8); Klebsiella aerogenes (n = 2); Serratia marcescens (n = 1); Morganella morganii (n = 2). To appraise antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) assays were run at different SC‐19 concentrations. Results When using SC‐19, S. aureus strains showed less bacterial growth, but no bactericidal effect was observed. The MIC and MBC of SC‐19 were 62.5 and 2000 μg/ml against S. aureus and were >2000 μg/ml against the other isolates obtained from OE, respectively. In addition, the MBICs and MBECs of SC‐19 against S. aureus were 125 and >2000 μg/ml, respectively. Conclusion Nowadays the acquired antibiotic resistance phenomenon has stimulated research into novel and more efficient therapeutic agents. Hence, we report that, helped by the structural diversity fostered herein by a range of bis(pyrazolyl)methane derivatives, SC‐19 can be a promising alternative therapeutic option for treating OE caused by S. aureus given the observed effects on both planktonic state and biofilm. Level of Evidence IV

Abstract The food enzyme α‐amylase (4‐α‐d‐glucan glucanhydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain NZYM‐AY by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in starch processing for the production of glucose syrup and other starch hydrolysates, and distilled alcohol production. Since residual amounts of total organic solids are removed by distillation and by the purification steps applied during the production of glucose syrups, dietary exposure estimation was considered unnecessary. The production strain of the food enzyme fulfils the requirements for the qualified presumption of safety (QPS) approach to safety assessment. As no other concerns arising from the manufacturing process have been identified, the Panel considers that toxicological tests are not needed for the assessment of this food enzyme. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use (other than distilled alcohol production) the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme did not give rise to safety concerns under the intended conditions of use.

Abstract EFSA was asked by the European Commission to evaluate synthetic biology (SynBio) developments for agri‐food use in the near future and to determine whether or not they are expected to constitute potential new hazards/risks. Moreover, EFSA was requested to evaluate the adequacy of existing guidelines for risk assessment of SynBio and if updated guidance is needed. The scope of this Opinion covers food and feed risk assessment, the variety of microorganisms that can be used in the food/feed chain and the whole spectrum of techniques used in SynBio. This Opinion complements a previously adopted Opinion with the evaluation of existing guidelines for the microbial characterisation and environmental risk assessment of microorganisms obtained through SynBio. The present Opinion confirms that microbial SynBio applications for food and feed use, with the exception of xenobionts, could be ready in the European Union in the next decade. New hazards were identified related to the use or production of unusual and/or new‐to‐nature components. Fifteen cases were selected for evaluating the adequacy of existing guidelines. These were generally adequate for assessing the product, the production process, nutritional and toxicological safety, allergenicity, exposure and post‐market monitoring. The comparative approach and a safety assessment per se could be applied depending on the degree of familiarity of the SynBio organism/product with the non‐genetically modified counterparts. Updated guidance is recommended for: (i) bacteriophages, protists/microalgae, (ii) exposure to plant protection products and biostimulants, (iii) xenobionts and (iv) feed additives for insects as target species. Development of risk assessment tools is recommended for assessing nutritional value of biomasses, influence of microorganisms on the gut microbiome and the gut function, allergenic potential of new‐to‐nature proteins, impact of horizontal gene transfer and potential risks of living cell intake. A further development towards a strain‐driven risk assessment approach is recommended.

Abstract The food enzyme mucorpepsin (aspartic endopeptidase, EC 3.4.23.23) is produced with the non‐genetically modified microorganism Rhizomucor miehei strain MMR 164 by Takabio. The enzyme is chemically modified to produce a thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in milk processing for cheese production. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.98 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,320 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,300. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and five matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme chymosin (EC 3.4.23.4) is produced with the genetically modified Kluyveromyces lactis strain CIN by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. It is intended to be used in milk processing for cheese production and for the production of fermented milk products. Dietary exposure was estimated to be up to 0.73 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 1,300. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched for and four matches were found. The Panel considered that under the intended conditions of use the risk of allergic sensitisation and elicitation reactions by dietary exposure, although unlikely, cannot be excluded, particularly for individuals sensitised to cedar pollen allergens. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme chymosin (EC 3.4.23.4) is produced with the genetically modified Aspergillus niger strain DSM 29544 by Chr. Hansen. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in milk processing for cheese production and for production of fermented milk products. Based on the maximum use levels, dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.09 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 84.1 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure above 930. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with respiratory allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme mucorpepsin (aspartic endopeptidase, EC 3.4.23.23) is produced with the non‐genetically modified microorganism Rhizomucor miehei strain MMR 164. The enzyme is chemically modified by DuPont Nutrition Biosciences (now IFF) to produce a thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in milk processing for cheese production. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.98 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,320 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,300. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and five matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The expansion of fields related to probiotics, microbiome‐targeted interventions and an evolving landscape for implementation across policy, industry and end users, signifies an era of important clinical translational changes. Characteristics and perception of traditional probiotics stemmed from the historical long‐term use of fermented products. Although the distinction between probiotic microorganisms and fermentation‐associated microbes is important, it is often confused as not all fermented foods are probiotic supplements. Current innovation in area of biotechnology and bioinformatics is emerging outside of the classical definitions and new probiotics will emerge from novel sources, challenging scientific as well as regulatory instructions. At the same time, the search for individual and group microbiome signatures – biomarkers in order to predict disease incidence, progression and response to treatment is a key area of microbiological and multidisciplinary research, enabled by efficient and powerful processing of large data sets. However, the regulation of marketed beneficial microbes and probiotics differs among countries and the basic level of classification, which depend on probiotic classification is not globally harmonised. At the same time, the regulation is very demanding to evaluate the safety of products on the market, so that only those products with scientific evidence benefits can obtain positive recognition in ways of health claims. Collaborative experimental and theoretical approaches and case studies have assisted the progress in this crosscutting area of research. There is a requirement to clearly specify criteria and provide details about ways and approaches of achieving those criteria with the intention that manufacturers can benefit from a transparent way of communicating product quality to end users.

Abstract The EFSA Panel on Food Additives and Flavourings (FAF Panel) provides a scientific opinion on the safety of a proposed amendment of the specifications of enzymatically produced steviol glycosides (E 960c) with respect to the inclusion of rebaudioside D produced via enzyme‐catalysed bioconversion of purified stevia leaf extract. Rebaudioside D (95% on dry basis) is produced via enzymatic bioconversion of purified stevia leaf extract using uridine diphosphate (UDP)‐glucosyltransferase (UGT) and sucrose synthase enzymes produced by the genetically modified yeast K. phaffii UGT‐A, that facilitates the transfer of glucose to purified stevia leaf extract via glycosidic bonds. The same enzymes from K. phaffii UGT‐A may be used in the manufacturing process of the food additive, rebaudioside M produced via enzyme modification of steviol glycosides from stevia (E 960c(i)). The Panel considered that separate specifications would be needed for this food additive produced via the manufacturing process described in the current application, aligned with those already established for E 960c(i). The Panel concluded that there is no toxicological concern for Rebaudioside D produced via enzymatic bioconversion of purified stevia leaf extract using UDP‐glucosyltransferase and sucrose synthase produced by a genetically modified strain of the yeast K. phaffii. However, based on the available data, the Panel could not exclude the possibility that some residual amount of DNA coding for the kanamycin resistance gene could remain in the final product. Should this gene propagate in microbiota due to the presence of recombinant DNA in the final product, this would be of concern. Therefore, the Panel concluded that the safety of Rebaudioside D produced via this enzymatic bioconversion was not sufficiently demonstrated with the available data given that the absence of recombinant DNA was not shown.

Abstract The food enzyme α‐glucosidase (α‐d‐glucoside glucohydrolase; EC 3.2.1.20) is produced with the non‐genetically modified Aspergillus niger strain AE‐TGU by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in baking processes, cereal‐based processes, brewing processes and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was only calculated for the remaining three food processes. Based on the maximum use levels recommended, dietary exposure was estimated to be up to 0.64 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,062 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 1,650. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme endo‐polygalacturonase ((1→4)‐α‐d‐galacturonan glycanohydrolase; EC 2.3.1.15), is produced with the genetically modified Aspergillus luchuensis strain FLYSC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in fruit and vegetable processing for juice production. Based on the maximum use level, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.138 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 800 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 5,800. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and six matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded for individuals sensitised to cedar or grass pollen or maize. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase, EC 3.2.1.3) is produced with the genetically modified Aspergillus niger strain NZYM‐BR by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in starch processing for the production of glucose syrups and distilled alcohol. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups and by distillation, dietary exposure estimation was considered not necessary. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,135 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched for and two matches were found. The Panel considered that under the intended conditions of use (other than distilled alcohol production) the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract Background Little information is available about endotoxemia in donkeys. Characterizing the systemic inflammatory response (SIRS) to lipopolysaccharide (LPS) in donkeys would provide valuable clinical and therapeutic information. The effects of meloxicam on endotoxemia have not been studied in this species. Objectives To study the pathophysiology and gene expression associated with experimentally induced endotoxemia, and evaluate the effects of meloxicam on experimentally induced endotoxemia in donkeys and in equine monocyte cultures. Animals Six healthy adult female donkeys. Methods Endotoxemia was induced by an IV infusion of LPS for 30 minutes. Animals either received 20 mL of saline or 0.6 mg/kg of meloxicam IV after LPS infusion. The experiments lasted 6 hours. Blood samples were collected serially for hematology, serum biochemistry, interleukin measurement, and leukocyte gene expression analysis. Vital signs were recorded throughout the study. Monocyte cultures were used to test the effects of meloxicam on LPS‐activated monocytes. Results Lipopolysaccharide induced fever, leukopenia, and neutropenia of similar magnitude in both groups, but meloxicam attenuated increases in plasma lactate, tumor necrosis factor‐alpha (TNFα), and interleukin 1β concentrations compared to controls. No differences were detected between groups for cytokine mRNA expression. Furthermore, meloxicam decreased TNFα release in LPS‐activated monocyte cultures. Conclusions and Clinical Importance Meloxicam could be a feasible option for the treatment of endotoxemia and SIRS in donkeys. Additional studies are necessary to investigate possible meloxicam‐related posttranscriptional regulation and to compare this drug with other nonsteroidal anti‐inflammatory drugs (NSAIDs) in animals with endotoxemia.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer‐associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2‐derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha‐smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR‐27a and let‐7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR‐27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR‐27a in canine fibroblasts and involved in their activation. Importantly, miR‐27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin‐6 (IL‐6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR‐27a may play an important role in the tumour–microenvironment crosstalk.

Abstract Aims Concerns about the prognostic value of NYHA functional class (FC) in heart failure (HF) patients carrying a prophylactic implantable cardioverter defibrillator (ICD) are still present. We aimed to compare whether mortality and arrhythmic risk were different, in a cohort of HF patients undergoing ICD‐only implant, according to their FC. Methods and results HF patients with left ventricle ejection fraction (LVEF) ≤35%, undergoing first prophylactic ICD‐only implant were collected from a multicentre nationwide registry (2006–2015). Six hundred and twenty‐one patients were identified (101 patients in NYHA I; 411 in NYHA II; 109 in NYHA III). After a mean follow‐up of 4.4 years (±2.1), 126 patients died (20.3%). All‐cause mortality risk was higher in symptomatic patients: 13.9% in NYHA I patients, 18.3% in NYHA II patients (HR: 1.8, 95% CI 1.1–3.2), and 32.9% in NYHA III patients (HR: 3.9, 95% CI 2.1–7.3). Seventy‐eight out of all deaths were due to cardiovascular causes (12.6%). Cardiovascular mortality risk was also higher in symptomatic patients: 6.9% in NYHA I patients, 11% in NYHA II patients (HR: 2.2, 95% CI 1.1–4.9), and 23.9% in NYHA III (HR: 5.5, 95% CI 2.4–12.7). One hundred and seventeen patients received a first appropriate ICD therapy (19.4%). Arrhythmia free survival did not differ among study groups [20.8% in NYHA I patients, 18.7% in NYHA II (HR: 1.1, 95% CI 0.6–1.7) and 20.8% in NYHA III patients (HR: 1.3, 95% CI 0.7–2.5)]. NYHA class independently predicted cardiovascular mortality (NYHA III vs. NYHA I: HR, 4.7; 95% CI, 1.7–12.8, P = 0.002; NYHA II vs. NYHA I: HR, 2.1, 95% CI, 1.0–5.6, P = 0.05) but not all‐cause death (NYHA III vs. NYHA I: HR: 1.8, 95% CI 0.8–3.9, P = 0.11; NYHA II vs. NYHA I: HR, 1.1, 95% CI 0.6–2.2, P = 0.71;). Atrial fibrillation, chronic kidney disease, and diabetes emerged as predictors of both all‐cause death [(HR: 1.8, 95% CI 1.2–2.8, P = 0.005), (HR: 2.2, 95% CI 1.4–3.4, P < 0.001), (HR: 2.0, 95% CI 1.3–3.1, P = 0.001), respectively] and cardiovascular mortality [(HR: 1.8, 95% CI 1.1–3.1, P = 0.02), (HR: 3.1, 95% CI 1.8–5.4, P < 0.001), (HR: 1.7, 95% CI 1.1–3, P = 0.032), respectively]. Conclusions Mortality in HF patients undergoing prophylactic ICD implantation was higher in symptomatic patients. NYHA functional class along with other comorbidities might be helpful to identify a subgroup of ICD carriers with poorer prognosis and higher risk of cardiovascular death.

Abstract The EFSA Panel on Food Additive and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. Glucosylated steviol glycosides consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin‐free, basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non‐genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyses the transfer of glucose from starch to steviol glycosides mixtures isolated from the dried leaves of Stevia Rebaudiana. The Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960) were considered to support their safety as food additive. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight (bw) per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended some modifications to the specifications proposed by the applicant for glucosylated steviol glycosides with respect to the assay, the definition of the proposed new food additive and the proposed maximum limits for arsenic.

Abstract The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase (cyclising), EC 2.4.1.19) is produced with Anoxybacillus caldiproteolyticus strain St‐88 by PureCircle USA. It is intended to be used in the manufacture of glycosylated steviol glycosides. Residual amounts of total organic solids are removed by the purification steps applied during the production of the modified steviol glycosides; consequently, dietary exposure was not calculated. For the same reason, toxicological studies other than assessment of allergenicity were not considered necessary. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme rennet containing chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1) is prepared from the abomasum (stomach) of suckling lambs, by Productos Nievi, SA. The food enzyme is intended to be used in milk processing for cheese production. As no concerns arise from the animal source of the food enzyme, from its manufacture, and based on the history of safe use and consumption, the Panel considered that toxicological data were not required and no exposure assessment was necessary. On the basis of literature data, the Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure could not be excluded, but the likelihood for this to occur was considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

While Tanzania is among the high TB burden countries to reach the WHO’s End TB 2030 milestones, 41% of the people estimated to have had TB in 2020 were not diagnosed and notified. As part of the response to close the TB treatment coverage gap, SHDEPHA+ Kahama conducted a TB REACH active case-finding (ACF) intervention among rural and mining communities in Northwest Tanzania to increase TB/HIV case notification from July 2017 to June 2020. The intervention successfully linked marginalized mining communities with integrated TB/HIV screening, diagnostic, and referral services, screening 144,707 people for TB of whom 24,200 were tested for TB and 4,478 were tested for HIV, diagnosing 1,499 people with TB and 1,273 people with HIV (including at least 154 people with TB/HIV coinfection). The intervention revealed that community-based ACF can ensure high rates of linkage to care among hard-to-reach populations for TB. Providing integrated TB and HIV screening and diagnostic services during evening hours (Moonlight Events) in and around mining settlements can yield a large number of people with undiagnosed TB and HIV. For TB, this is true not only amongst miners but also FSW living in the same communities, who appear to be at similar or equally high risk of infection. Local NGOs can help to bridge the TB treatment coverage gap and to improve TB and HIV health outcomes by linking these marginalized groups with public sector services. Capturing the number of referrals arriving at CTCs is an important next step to identify how well the integrated TB/HIV outreach services operate and how they can be strengthened.

Abstract The food enzyme rennet paste containing chymosin (EC 3.4.23.4), pepsin A (EC 3.4.23.1) and triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is prepared from the abomasum of suckling goats, lambs and calves by Caglificio Clerici S.p.A. The food enzyme is intended to be used in milk processing for cheese production. As no concerns arise from the animal source of the food enzyme, from its manufacture, and based on the history of safe use and consumption, the Panel considers that toxicological data were not required and no exposure assessment was necessary. On the basis of literature data, the Panel considers that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure could not be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme isoamylase (glycogen α‐1,6‐glucanohydrolase, EC 3.2.1.68) is produced with Dyella sp. strain MU 1174 by Hayashibara Co. Ltd. In a previous opinion, the Panel on Food Contact Materials, Enzymes and Processing Aids of EFSA could not conclude on the safety of this food enzyme due to uncertainties about the presence of a gene conferring resistance to antimicrobials in the genome of the production strain and its potential transfer to the food enzyme. New whole genome sequence data provided by the applicant showed that the production strain Dyella sp. MU 1174 does not contain antimicrobial resistance genes of concern. Based on the new data provided and the evaluation of the data previously submitted, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA developed an updated scientific guidance to assist applicants in the preparation of applications for food enzymes. This guidance describes the scientific data to be included in applications for the authorisation of food enzymes, as well as for the extension of use for existing authorisations, in accordance with Regulation (EC) No 1331/2008 and its implementing rules. Information to be provided in applications relates to source, production and characteristics of the food enzyme, toxicological data, allergenicity and dietary exposure estimation. Source, production and characteristics of the food enzyme are first considered only for enzymes of microbial origin and subsequently for those enzymes derived from plants and for enzymes from animal sources. Finally, the data requested for toxicology, allergenicity and dietary exposure applies to all food enzymes independent of the source. On the basis of the submitted data, EFSA will assess the safety of food enzymes and conclude whether or not they present a risk to human health under the proposed conditions of use.

Type 2 diabetes mellitus (T2DM) has been associated with an increased risk of cancer, including colon cancer (CC). However, we recently reported no influence of T2DM on CC prognosis, suggesting that any effect might be at the early stages of tumor development. We hypothesized that T2DM may create an environment in the healthy tissue, which acts as a carcinogenesis driver in agreement with the field of cancerization concept. Here, we focused on early carcinogenesis by analyzing paired tumor and normal colonic mucosa samples from the same patients. The proteome of CC and paired mucosa was quantitatively analyzed in 28 individuals (12 diabetics and 16 nondiabetics) by mass spectrometry with isobaric labeling. Out of 3076 identified proteins, 425 were differentially expressed at the tumor in diabetics compared with nondiabetics. In the adjacent mucosa, 143 proteins were differentially expressed in diabetics and nondiabetics. An enrichment analysis of this signature pointed to mitochondria, ribosome, and translation. Only six proteins were upregulated by diabetes both in tumor and mucosa, of which five were mitochondrial proteins. Differential expression in diabetic versus nondiabetic mucosa was confirmed for MRPL53, MRPL18, and TIMM8B. Higher levels of MRPL18, TIMM8B, and EIF1A were also found in normal colon epithelial cells exposed to high‐glucose conditions. We conclude that T2DM is associated with specific molecular changes in the normal mucosa of CC patients, consistent with field of cancerization in a diabetic environment. The mitochondrial protein signature identifies a potential therapeutic target that could underlie the higher risk of CC in diabetics.

The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. While several clinical studies show a higher incidence of CC and a lower survival rate in diabetics, others report no association. Our own experience indicates that diabetes does not seem to worsen the prognosis once the tumor is present. Despite this controversy, there are no wide‐spectrum molecular studies that delve into the impact of T2DM‐related mechanisms in colon carcinogenesis. Here, we present a transcriptomic and proteomic profiling of paired tumor and normal colon mucosa samples in a cohort of 42 CC patients, 23 of which have T2DM. We used gene set enrichment and network approaches to extract relevant pathways in diabetics, referenced them to current knowledge, and tested them using in vitro techniques. Through our transcriptomics approach, we identified an unexpected overlap of pathways overrepresented in diabetics compared to nondiabetics, in both tumor and normal mucosa, including diabetes‐related metabolic and signaling processes. Proteomic approaches highlighted several cancer‐related signaling routes in diabetics found only in normal mucosa, not in tumors. An integration of the transcriptome and proteome analyses suggested the deregulation of key pathways related to colon carcinogenesis which converged on tumor initiation axis TEAD/YAP‐TAZ as a potential initiator of the process. In vitro studies confirmed upregulation of this pathway in nontumor colon cells under high‐glucose conditions. In conclusion, T2DM associates with deregulation of cancer‐related processes in normal colon mucosa adjacent to tissue which has undergone a malignant transformation. These data support that in diabetic patients, the local microenvironment in normal colon mucosa may be a factor driving field cancerization promoting carcinogenesis. Our results set a new framework to study links between diabetes and colon cancer, including a new role of the TEAD/YAP‐TAZ complex as a potential driver.

Background The determinants and consequences of pulmonary hypertension after successfully corrected valvular heart disease remain poorly understood. We aim to clarify the hemodynamic bases and risk factors for mortality in patients with this condition. Methods and Results We analyzed long‐term follow‐up data of 222 patients with pulmonary hypertension and valvular heart disease successfully corrected at least 1 year before enrollment who had undergone comprehensive hemodynamic and imaging characterization as per the SIOVAC (Sildenafil for Improving Outcomes After Valvular Correction) clinical trial. Median (interquartile range) mean pulmonary pressure was 37 mm Hg (32–44 mm Hg) and pulmonary artery wedge pressure was 23 mm Hg (18–26 mm Hg). Most patients were classified either as having combined precapillary and postcapillary or isolated postcapillary pulmonary hypertension. After a median follow‐up of 4.5 years, 91 deaths accounted for 4.21 higher‐than‐expected mortality in the age‐matched population. Risk factors for mortality were male sex, older age, diabetes mellitus, World Health Organization functional class III and higher pulmonary vascular resistance—either measured by catheterization or approximated from ultrasound data. Higher pulmonary vascular resistance was related to diabetes mellitus and smaller residual aortic and mitral valve areas. In turn, the latter correlated with prosthetic nominal size. Six‐month changes in the composite clinical score and in the 6‐minute walk test distance were related to survival. Conclusions Persistent valvular heart disease–pulmonary hypertension is an ominous disease that is almost universally associated with elevated pulmonary artery wedge pressure. Pulmonary vascular resistance is a major determinant of mortality in this condition and is related to diabetes mellitus and the residual effective area of the corrected valve. These findings have important implications for individualizing valve correction procedures. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT00862043.

Abstract Epidemiological studies comparing clinical and commensal Staphylococcus epidermidis isolates suggest that biofilm formation is a discriminant biomarker. A study showed that four non‐biofilm‐forming clinical S. epidermidis isolates could form an induced biofilm by trypsin treatment, suggesting that S. epidermidis can form biofilms in a protease‐independent way and in a trypsin‐induced way. In this study, the trypsin capacity to induce biofilm formation was evaluated in non‐biofilm‐forming S. epidermidis isolates (n = 133) in order to support this mechanism and to establish the importance of total biofilms (meaning the sum of protease‐independent biofilm and trypsin‐induced biofilm). Staphylococcus epidermidis isolates from ocular infections (OI; n = 24), prosthetic joint infections (PJI; n = 64), and healthy skin (HS‐1; n = 100) were screened for protease‐independent biofilm formation according to Christensen's method. The result was that there are significant differences (p

Abstract Objectives The addition of RANKL/RANK blockade to immune checkpoint inhibitors (ICIs) such as anti‐PD‐1/PD‐L1 and anti‐CTLA4 antibodies is associated with increased anti‐tumor immunity in mice. Recent retrospective clinical studies in patients with advanced melanoma and lung cancer suggest the addition of anti‐RANKL antibody to ICI increases the overall response rate relative to ICI treatment alone. Based on this rationale, we developed a novel bispecific antibody (BsAb) co‐targeting RANKL and PD‐1. Methods We characterized target binding and functional activity of the anti‐RANKL/PD‐1 BsAb in cell‐based assays. Anti‐tumor activity was confirmed in experimental lung metastasis models and in mice with established subcutaneously transplanted tumors. Results The anti‐RANKL/PD‐1 BsAb retained binding to both RANKL and PD‐1 and blocked the interaction with respective counter‐structures RANK and PD‐L1. The inhibitory effect of anti‐RANKL/PD‐1 BsAb was confirmed by demonstrating a complete block of RANKL‐dependent osteoclast formation. Monotherapy activity of anti‐RANKL/PD‐1 BsAb was observed in anti‐PD‐1 resistant tumors and, when combined with anti‐CTLA‐4 mAb, increased anti‐tumor responses. An equivalent or superior anti‐tumor response was observed with the anti‐RANKL/PD‐1 BsAb compared with the combination of parental anti‐RANKL plus anti‐PD‐1 antibodies depending upon the tumor model. Discussion Mechanistically, the anti‐tumor activity of anti‐RANKL/PD‐1 BsAb required CD8+T cells, host PD‐1 and IFNγ. Targeting RANKL and PD‐1 simultaneously within the tumor microenvironment (TME) improved anti‐tumor efficacy compared with combination of two separate mAbs. Conclusion In summary, the bispecific anti‐RANKL/PD‐1 antibody demonstrates potent tumor growth inhibition in settings of ICI resistance and represents a novel modality for clinical development in advanced cancer.

Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction.

At the northwestern edge of South America is located Ecuador. This place is a classical example of an active continental margin with widespread active volcanism. Detailed studies about the impact of volcanic ash on human health are still lacking. Therefore, the disease of exposed populations is unknown. The objective of the present investigation was to assess the biological impact of Pichincha volcanic ash on cell culture and inflammation in murine lung tissues that will contribute to the understanding of the hazards. In this study, the in vivo phase was performed in mice C57BL/6 exposed to several doses of volcanic ash (0.5, 1, and 3.75 mg/100 g mouse body weight). The body weight and survival were controlled during seven days of treatment. The expression of inflammation markers NRLP 3, caspase-1, pro-IL-1, IL-1β, IL-6, IL-8, and h-HPRT was analyzed. The in vitro phase was performed in lung cancer cells A549, peritoneal macrophages, and McCoy cells exposing them to different concentrations of volcanic ash (80, 320, and 1280 μg/cm3) to determine the cytotoxicity and the production of reactive oxygen species. The ash initiated activation of the inflammasome complex NRLP 3 and the initiation of a proinflammatory activity in the murine lung tissue depending on the concentration of this agent. The viability of A549 and McCoy cell decreased with the length of exposure and increased with the concentration of volcanic ash. The activity in superoxide dismutase decreased by about 60%, leading to the formation of reactive oxygen species. These results associated with compounds contained in Pichincha volcanic ash are considered hazardous elements which induce inflammation leading to activate inflammasome NRLP, releasing reactive oxygen species, and producing changes in cell morphology and density, all of which are expression of cytotoxicity.

Abstract Beltherm MP/ML is a feed additive that contains endo‐1,4‐beta‐xylanase to be used as a zootechnical additive in poultry species. The production strain of the xylanase is a genetically modified strain of Bacillus subtilis. This species is considered by EFSA to qualify for the qualified presumption of safety (QPS) approach to safety assessment. The strain was unequivocally identified and qualifications were met. The genetic modification along with the results on the investigations regarding the presence? of the production strain and its DNA in the product were evaluated by the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) and raised no safety concerns. The FEEDAP Panel also concluded that the additive is safe for chickens and turkeys for fattening, chickens reared for laying, turkeys reared for breeding, turkeys for breeding purposes and minor poultry species at 100 ADXU/kg feed with a wide margin of safety. The Panel also concluded that no toxicological concerns for the consumer would arise from the use of Beltherm as a feed additive. The additive is considered a potential respiratory sensitiser for users. The Panel concluded that the use of the product as a feed additive raises no safety concerns for the environment. The additive has the potential to improve the performance of chickens for fattening, chickens reared for laying and minor growing poultry species at the dose of 100 ADXU/kg feed. The Panel could not conclude on the efficacy of the product in turkeys for fattening or reared for breeding or in turkeys for breeding purposes.

Abstract The product subject of this assessment is l‐threonine produced by fermentation with a genetically modified strain of Escherichia coli (CGMCC 11473). It is intended to be used in feed and water for drinking for all animal species and categories. It was not possible to characterise the genetic modification with the information provided. Uncertainty remained on the possible presence of cells from the production strain and their recombinant DNA in the product. Therefore, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) could not conclude on the safety of the product l‐threonine, produced by fermentation with Escherichia coli CGMCC 11473 for the target species, consumers and the environment. The FEEDAP Panel has concerns on the safety of the simultaneous oral administration of threonine containing additives via water for drinking and feed. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be an irritant for skin and eyes or to be a skin sensitiser. There is a risk from the exposure by inhalation to endotoxins for persons handling the additive. The product under assessment is considered an efficacious source of the amino acid l‐threonine for all animal species. For l‐threonine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.

Analyses of calcium compounds in cladodes, soluble dietary fiber (SDF), and insoluble dietary fiber (IDF) of Opuntia ficus indica are reported. The characterization of calcium compounds was performed by using Scanning Electron Microscopy, Energy Dispersive Spectrometry, X-ray diffraction, and infrared spectroscopy. Atomic Absorption Spectroscopy and titrimetric methods were used for quantification of total calcium and calcium compounds. Whewellite (CaC2O4·H2O), weddellite (CaC2O4·(H2O)2.375), and calcite (CaCO3) were identified in all samples. Significant differences (P≤0.05) in the total calcium contents were detected between samples. CaC2O4·H2O content in cladodes and IDF was significantly higher (P≤0.05) in comparison to that observed in SDF, whereas minimum concentration of CaCO3 was detected in IDF with regard to CaCO3 contents observed in cladodes and SDF. Additionally, molar ratio oxalate : Ca2+ in all samples changed in a range from 0.03 to 0.23. These results support that calcium bioavailability in O. ficus indica modifies according to calcium compounds distribution.

A twin pregnancy with a complete hydatidiform mole and a coexisting normal fetus (CHMF) is a rare clinical scenario, and it carries many associated pregnancy and postnatal risks. Limited numbers of case studies exist reporting an outcome of live birth, and only three prior cases report the presentation of a hydatidiform mole as a placenta previa. We report a case of CHMF with the molar component presenting antenatally as a placenta previa, which ultimately resulted in placenta accreta at the time of delivery. A live male infant was delivered at 34 weeks’ gestation via planned cesarean section, and a hysterectomy was performed following unsuccessful removal of the molar component. We additionally utilized previously described methods of placing internal iliac balloons and ureteral stents prior to delivery. In such a high-risk pregnancy with a known molar previa component, these surgical preparation measures may be of benefit.

In pancreatic ductal adenocarcinoma (PDAC), metabolic rewiring and resistance to standard therapy are closely associated. PDAC cells show enormous requirements for glucose‐derived citrate, the first rate‐limiting metabolite in the synthesis of new lipids. Both the expression and activity of citrate synthase (CS) are extraordinarily upregulated in PDAC. However, no previous relationship between gemcitabine response and citrate metabolism has been documented in pancreatic cancer. Here, we report for the first time that pharmacological doses of vitamin C are capable of exerting an inhibitory action on the activity of CS, reducing glucose‐derived citrate levels. Moreover, ascorbate targets citrate metabolism towards the de novo lipogenesis pathway, impairing fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression. Lowered citrate availability was found to be directly associated with diminished proliferation and, remarkably, enhanced gemcitabine response. Moreover, the deregulated citrate‐derived lipogenic pathway correlated with a remarkable decrease in extracellular pH through inhibition of lactate dehydrogenase (LDH) and overall reduced glycolytic metabolism. Modulation of citric acid metabolism in highly chemoresistant pancreatic adenocarcinoma, through molecules such as vitamin C, could be considered as a future clinical option to improve patient response to standard chemotherapy regimens.

Abstract Background Little information exists about vitamin D status in bitches with mammary tumors. Objectives To determine whether low plasma vitamin D concentrations are found in bitches with mammary tumors. Animals Eighty‐five client‐owned bitches with mammary tumors (n = 21 benign, n = 64 malignant) and 39 age‐matched healthy bitches. Methods Case‐control study. Plasma ionized and total calcium, phosphorus, magnesium, urea, creatinine, albumin, total proteins, alanine aminotransferase, alkaline phosphatase, parathyroid hormone (PTH), calcitriol (1,25‐dihydroxyvitamin D), and 25‐hydroxyvitamin D concentrations were measured in all bitches at the time of clinical diagnosis and before any treatments. Statistical analysis was performed to compare variables among groups (control, benign, and malignant). Results No significant differences were found when plasma 25‐hydroxyvitamin D concentrations in bitches with malignant (148.9 [59.9] ng/mL) and benign mammary tumors (150.1 [122.3] ng/mL) were compared with control group (129.9 [54.5] ng/mL). Parathyroid hormone was significantly higher in bitches with malignant (19.9 [20.5] pg/mL), and benign mammary tumors (14.6 [14.9] pg/mL) compared with control group (7.5 [7.5] pg/mL; P

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 the safety of an extension of use of oil from Schizochytrium limacinum (strain FCC‐3204) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The extension of use pertains to the use of the NF as a food ingredient in protein products at a maximum use level of 1 g of docosahexaenoic acid (DHA) in 100 g of product. The Panel considers that the information provided on the composition and the production process is sufficiently described and does not raise safety concerns. S. limacinum was attributed the qualified presumption of safety (QPS) status with the qualification ‘for production purposes only’. Data provided demonstrated the absence of viable cells in the NF. Under the proposed extension of use, the highest intake estimate (at the 95th percentile) of DHA from the NF in protein products is 6.3 mg DHA/kg bw per day for adolescents. The Panel notes that the exposure to DHA from the new intended use of the NF in protein products is very low compared to the exposure to DHA from the already authorised food categories (excluding food supplements). The Panel concludes that the NF (oil from S. limacinum (FCC‐3204)) is safe under the new intended use.

Abstract The food enzyme, a triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3), is produced with the non‐genetically modified Limtongozyma cylindracea strain AE‐LAYH (B) by Amano Enzyme Inc. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 0.315 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the quality presumption of safety (QPS) approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A homology search for the amino acid sequence of the food enzyme to those of known allergens was made and one match with a honeybee venom allergen was found. The Panel considered that a risk of allergic reactions by dietary exposure, particularly in individuals allergic to honey, cannot be excluded, but is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

ABSTRACT Background Recurrent squamous cell carcinoma (SCC) of the head and neck (SCCHN) remains a formidable clinical challenge despite available treatments. The phosphatidylinositol 3‐kinase (PI3K) pathway has been identified as a potential therapeutic target, and alpelisib, a selective PI3Kα inhibitor, has demonstrated efficacy in certain malignancies. Combining this targeted therapy with immunotherapy has been suggested in previous studies as a promising strategy to bolster the immune response against cancer. Cases A 69‐year‐old woman with locoregional recurrence of PIK3CA‐mutated SCC of the left maxilla and cervical nodal metastases. Several chemotherapeutic regimens, including cisplatin, docetaxel, 5FU, chemoradiotherapy, and mono‐immunotherapy, resulted in disease progression. Alpelisib combined with pembrolizumab led to a sustained response for 9 months. A 58‐year‐old man with recurrent metastatic PIK3CA‐mutated SCC of the oropharynx, involving the left lung, hilar, and mediastinal lymph nodes. Despite prior palliative radiation and platinum‐based chemotherapy with pembrolizumab and cetuximab, treatment with alpelisib and nivolumab resulted in a partial response. Severe hyperglycemia and rash led to treatment discontinuation. Conclusion Our findings highlight the potential of this innovative therapeutic combination, suggesting a need for further investigations in this setting.

Abstract The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of curdlan as a new food additive used as firming and gelling agent, stabiliser, thickener. Curdlan is a high molecular weight polysaccharide consisting of β‐1,3‐linked glucose units, produced by fermentation from Rhizobium radiobacter biovar 1 strain NTK‐u. The toxicological dataset consisted of sub‐chronic, chronic and carcinogenicity, reproductive and developmental toxicity studies as well as genotoxicity. In vivo data showed that curdlan is not absorbed as such but is extensively metabolised by the gut microbiota into CO2 and other innocuous compounds. Curdlan was not genotoxic and was well‐tolerated with no overt organ‐specific toxicity. Effects observed at very high doses of curdlan, such as decreased growth and increased cecum weight, are common for indigestible bulking compounds and therefore considered physiological responses. In a combined three‐generation reproductive and developmental toxicity study, decreased pup weight was observed during lactation at 7500 mg curdlan/kg body weight (bw) per day, the highest dose tested. The Panel considered the observed effects as treatment‐related and adverse, although likely secondary to nutritional imbalance and identified a conservative no observed adverse effect level (NOAEL) of 2500 mg/kg bw per day. Despite the limitations noted in the dataset, the Panel was able to conclude applying the margin of exposure (MOE) approach. Given that curdlan and its break‐down products are not absorbed and that the identified adverse effect is neither systemic nor local, no adjustment factor was deemed necessary. Thus, an MOE of at least 1 was considered sufficient. The highest exposure estimate was 1441 mg/kg bw per day in toddlers at the 95th percentile of the proposed maximum use level exposure assessment scenario. The Panel concluded that there is no safety concern for the use of curdlan as a food additive at the proposed uses and use levels.

Abstract The European Commission requested EFSA to update the scientific guidance for the preparation of applications for authorisation of novel foods, previously developed following the adoption of Regulation (EU) 2015/2283 on novel foods. This guidance document provides advice on the scientific information needed to be submitted by the applicant towards demonstrating the safety of the novel food. Requirements pertain to the description of the novel food, production process, compositional data, specifications, proposed uses and use levels and anticipated intake of the novel food. Furthermore, information needed in sections on the history of use of the novel food and/or its source, absorption, distribution, metabolism, excretion, toxicological information, nutritional information and allergenicity is also described. The applicant should integrate and interpret the data presented in the different sections to provide their overall considerations on how the information supports the safety of the novel food under the proposed conditions of use. Where potential health hazards have been identified, they are to be discussed in relation to the anticipated intake of the novel food and the proposed target populations. On the basis of the information provided, EFSA will assess the safety of the novel food under the proposed conditions of use.

Abstract Background Intervertebral disc herniation is widely recognized as the most common cause of myelopathy in dogs older than 2 years; however, the prevalence of various causes of myelopathy in younger dogs has not been reported. Hypothesis/Objectives To describe the prevalence, clinical presentation, and etiology of myelopathy in dogs aged 18 months or less. Secondarily, to investigate which clinical features were associated with each of the most common etiologies. Animals Two hundred twenty‐four dogs aged 18 months or less with myelopathy were included in the study. Methods Retrospective review of clinical records from 4 referral institutions. Multivariable logistic regression analyses assessed which clinical features were associated with each diagnosis. Results French bulldogs (n = 51, 22.8%), pugs (n = 18, 8.0%), crossbreeds (n = 12, 5.4%), and English bulldogs (n = 11, 4.9%) were the most frequently affected breeds. Overall, 31 diagnoses were reached. The 5 most frequent diagnoses were vertebral malformation (VM; n = 42, 18.8%), spinal arachnoid diverticulum (SAD; n = 28, 12.5%), traumatic fracture of the vertebral column (n = 22, 9.8%), atlantoaxial instability (n = 18, 8.0%), and osseous‐associated cervical spondylomyelopathy (n = 17, 7.6%). Intervertebral disc extrusion (IVDE) accounted for 4.5% of cases (n = 10). A final diagnosis of VM was associated with younger, screw‐tailed, and pug breeds with chronic signs of T3‐L3 myelopathy. SAD was associated with screw‐tailed and pug breeds with nonpainful clinical signs. Intervertebral disc extrusion was associated with older, screw‐tailed, and pug breeds with shorter duration of clinical signs. Conclusions and Clinical Importance Prioritization of differential diagnoses for dogs presenting with signs of myelopathy when aged 18 months or less should differ to those for older dogs, with IVDE not the most common cause in the former.

Abstract There are limited data on first seizure (FS) among adults in low and middle‐income countries. We describe findings from a prospective cohort study involving 180 adults presenting with seizures in emergency departments in five Latin American countries. Overall, 102 participants (56.7%) had acute symptomatic seizures (ASyS) while 78 (43.3%) had unprovoked seizures (UPS). Among patients with ASyS, 55 (53.9%) had structural causes, with stroke (n = 24, 23.5%), tumor (n = 10, 9.8%), and trauma (n = 3, 3%) being the most frequent. Nineteen patients (18.6%) had infectious causes, including four (4%) with meningoencephalitis, three (3%) neurocysticercosis, and two (2%) bacterial meningoencephalitis. Twenty patients (19.6%) had metabolic/toxic evidence, including four (4%) with uremic encephalopathy, two (2%) hyponatremia, and three (3%) acute alcohol intoxication. Immune dysfunction was present in seven (7%) patients and neurodegenerative in two (2%). Among participants with UPS, 45 (57.7%) had unknown etiology, 24 (30.7%) had evidence of structural disorders (remote symptomatic), four (5%) were related to infectious etiology (>7 days before the seizure), and five (6.4%) had genetic causes. During the 3‐ and 6‐month follow‐up, 29.8% and 14% of patients with UPS, respectively, experienced seizure recurrence, while 23.9% and 24.5% of patients with ASyS had seizure recurrence. Longer follow‐up is necessary to assess seizure recurrence for patients with ASyS after the acute cause is resolved and to determine the 10‐year risk of recurrence, which is part of the definition of epilepsy. Plain Language Summary We monitored 180 adults who presented with their first seizure in emergency departments across five Latin American countries. Among these patients, 57% had acute symptomatic seizures, with structural causes such as stroke (23%), infection (17%), or tumor (10%) being more prevalent. Among the 43% with unprovoked seizures, 58% showed no identifiable acute cause, while 6.4% were due to genetics. Within 3 months after their initial seizure, 26.6% of individuals experienced a second seizure, with 11.9% continuing to have seizures in Months 3–6. Between Months 3 and 6, an additional 20% of patients encountered a second seizure. Research is needed to better understand the cause and prognosis of these patients to improve outcomes.