Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange.

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

Implementation of state health insurance benefit mandates for cancer-related fertility preservation: following policy through a complex system.

BACKGROUND: A myriad of federal, state, and organizational policies are designed to improve access to evidence-based healthcare, but the impact of these policies likely varies due to contextual determinants of, reinterpretations of, and poor compliance with policy requirements throughout implementation. Strategies enhancing implementation and compliance with policy intent can improve population health. Critically assessing the multi-level environments where health policies and their related health services are implemented is essential to designing effective policy-level implementation strategies. California passed a 2019 health insurance benefit mandate requiring coverage of fertility preservation services for individuals at risk of infertility due to medical treatments, in order to improve access to services that are otherwise cost prohibitive. Our objective was to document and understand the multi-level environment, relationships, and activities involved in using state benefit mandates to facilitate patient access to fertility preservation services. METHODS: We conducted a mixed-methods study and used the policy-optimized exploration, preparation, implementation, and sustainment (EPIS) framework to analyze the implementation of California's fertility preservation benefit mandate (SB 600) at and between the state insurance regulator, insurer, and clinic levels. RESULTS: Seventeen publicly available fertility preservation benefit mandate-relevant documents were reviewed. Interviews were conducted with four insurers; 25 financial, administrative, and provider participants from 16 oncology and fertility clinics; three fertility pharmaceutical representatives; and two patient advocates. The mandate and insurance regulator guidance represented two "Big P" (system level) policies that gave rise to a host of "little p" (organizational) policies by and between the regulator, insurers, clinics, and patients. Many little p policies were bridging factors to support implementation across levels and fertility preservation service access. Characterizing the mandate's functions (i.e., policy goals) and forms (i.e., ways that policies were enacted) led to identification of (1) intended and unintended implementation, service, and patient outcomes, (2) implementation processes by level and EPIS phase, (3) actor-delineated key processes and heterogeneity among them, and (4) inner and outer context determinants that drove adaptations. CONCLUSIONS: Following the midstream and downstream implementation of a state health insurance benefit mandate, data generated will enable development of policy-level implementation strategies, evaluation of determinants and important outcomes of effective implementation, and design of future mandates to improve fit and fidelity.

The moon cycle influences reproductive and productive traits in guinea pigs (Cavia porcellus) from a tropical Andean area.

There is currently innumerable evidence showing that the lunar cycle affects various reproductive aspects in farm animals. However, there is very little information on the effect of the lunar cycle on productive traits in these species. A retrospective study was conducted (2015-2018) to evaluate the influence of the lunar cycle on some reproductive and productive traits in a guinea pig (Cavia porcellus) production system in the southern Andean region of Ecuador. A total of 7352 productive and reproductive records of guinea pig females housed in 3 m2 cages with a breeding male were analyzed. The following variables were considered: offspring sex ratio, litter size per cage, number of weaned guinea pigs, mortality, individual and litter weaning weight of guinea pigs, and calving frequency. The lunar cycle was split into eight periods of ~3.7-d length each. Data were analyzed by logistic regression and general linear model, and means were compared by the least mean squares method of the SAS. The offspring sex ratio was not correlated to the lunar cycle. The lunar cycle at mating and calving influenced the litter size per cage, number of weaned, and mortality of guinea pigs. The individual and litter-weaning weights were influenced by the lunar cycle at calving. The frequency of calving was greater around the new and full moon than in the remaining periods of the lunar cycle. In conclusion, the lunar cycle influenced several productive and reproductive traits in guinea pigs, such as litter size, mortality, number of pups weaned, and individual and litter weaning weights. This valuable information may have practical applications in management of guinea pig production systems.

Safety of lacto-N-fucopentaose I/2'-fucosyllactose (LNFP-I/2'-FL) mixture as a novel food pursuant to Regulation (EU) 2015/2283.

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 lacto-N-fucopentaose I (LNFP-I)/2'-fucosyllactose (2'-FL) mixture as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharides (HiMO) LNFP-I and 2'-FL, but it also contains d-lactose, lacto-N-tetraose, difucosyllactose, 3-fucosyllactose, LNFP-I fructose isomer, 2'-fucosyl-d-lactulose, l-fucose and 2'-fucosyl-d-lactitol, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli K-12 DH1 MDO MP2173b) of E. coli K-12 DH1 (DSM 4235). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF in a variety of foods, including infant formula (IF) and follow-on formula, foods for infants and toddlers, foods for special medical purposes and food supplements (FS). The target population is the general population. The anticipated daily intake of LNFP-I from use in IF is similar to the estimated natural mean highest daily intake in breastfed infants. Overall, the anticipated daily intake of LNFP-I from the NF as a food ingredient at the maximum proposed use levels is unlikely to exceed the intake level of breastfed infants on a body weight basis. The intake in breastfed infants on a body weight basis is expected to be safe also for other population groups. The anticipated 2'-FL intake is generally rather low. The use of the NF in FS is not intended if other foods with added NF components or human milk (for infants and young children) are consumed on the same day. The Panel concludes that the NF, a mixture of LNFP-I and 2'-FL, is safe under the proposed conditions of use.

Safety of 2'-fucosyllactose (2'-FL) produced by a derivative strain (Escherichia coli SGR5) of E. coli W (ATCC 9637) as a Novel Food pursuant to Regulation (EU) 2015/2283.

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 2'-fucosyllactose (2'-FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharide (HiMO) 2'-FL, but it also contains d-lactose, l-fucose, fucosylgalactose, difucosyllactose, d-glucose and d-galactose, and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli SGR5) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant applies for the same use and use levels as already authorised for 2'-FL and included in the Union list of NFs, with the general population as target population. The Panel noted that the available intake estimate is not recent (2015) and based on a different database (2008-2010 UK data) than that used by EFSA. For this reason, the Panel decided to perform a new intake estimate according to the current EFSA approach. The Panel notes that the highest P95 daily intake of the NF from the use as food ingredient is higher than the estimated natural highest mean daily intake in breastfed infants and marginally higher in young children. The applicant also proposes to extend the use of 2'-FL in food supplements (FS) for infants at the use level of 1.2 g/day. The resulting estimated intake in infants from the proposed use in FS is within the natural intake of 2'-FL in breastfed infants. FS are not intended to be used if other foods with added 2'-FL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Safety of the extension of use of 2'-fucosyllactose (2'-FL) as a novel food pursuant to Regulation (EU) 2015/2283.

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 the extension of use of 2'-fucosyllactose (2'-FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is already authorised as ingredient in several food categories, including infant formula (IF) and follow-on formula (FOF). The applicant proposed to increase the maximum use levels of the NF in IF and FOF. EFSA estimated the anticipated daily intake of the NF from the proposed extension of use, including the already authorised conditions of use in other food categories. Additionally, a new intake estimate limited to the already authorised conditions of use of the NF was carried out following EFSA's current approach. The estimated daily intake of the NF from high consumption of IF alone at the proposed maximum use level in infants < 16 weeks of age is similar to the estimated natural highest mean daily intake of 2'-FL from human milk in breastfed infants. The estimated highest P95 daily intakes of the NF from the proposed extension of use in IF and FOF (including the authorised uses in other food categories) and from the already authorised conditions of use, are comparable and both higher than the estimated natural highest mean daily intake of 2'-FL from human milk in infants and to a lesser extent in young children. The Panel considers that the proposed extension of use of the NF in IF and FOF only marginally affects the highest P95 daily intake estimate from the authorised conditions of use, and therefore does not affect the safety of the NF. The Panel concludes that the NF, 2'-FL, is safe under the proposed conditions of use.

Statement on how to interpret the QPS qualification on 'acquired antimicrobial resistance genes'.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case-by-case assessment is necessary.

Implementation of state health insurance benefit mandates for cancer-related fertility preservation: Following policy through a complex system.

Background: A myriad of federal, state, and organizational policies are designed to improve access to evidence-based healthcare, but the impact of these policies likely varies due to contextual determinants, re-interpretations of and poor compliance with policy requirements throughout implementation. Strategies enhancing implementation and compliance with policy intent can improve population health. Critically assessing the multi-level environments where health policies and their related health services are implemented is essential to designing effective policy-level implementation strategies. California passed a 2019 health insurance benefit mandate requiring coverage of fertility preservation (FP) services for individuals at risk of infertility due to medical treatments to improve access to services that are otherwise cost-prohibitive. Our objective was to document and understand multi-level environment, relationships, and activities involved in using state benefit mandates to facilitate patient access to FP services. Methods: We conducted a mixed-methods study and used the policy-optimized Exploration, Preparation, Implementation, Sustainment (EPIS) framework to analyze implementation of California's fertility preservation benefit mandate (SB 600) at and between the state insurance regulator, insurer and clinic levels. Results: Seventeen publicly available FP benefit mandate-relevant documents were reviewed, and four insurers, 25 financial, administrative and provider participants from 16 oncology and fertility clinics, three fertility pharmaceutical representatives, and two patient advocates were interviewed. The mandate and insurance regulator guidance represented two "Big P" (system level) policies that gave rise to a host of "little p" (organizational) policies by and between the regulator, insurers, clinics, and patients. Many little p policies were bridging factors to support implementation across levels and FP service access. Characterizing the mandate's functions (i.e., policy goals) and forms (i.e., ways that policies were enacted) led to identification of (1) intended and unintended implementation, service, and patient outcomes; (2) implementation processes by level, EPIS phase; (3) actor-delineated key processes and heterogeneity among them; and (4) inner and outer context determinants that drove adaptations. Conclusions: Following the mid- and down-stream implementation of a state health insurance benefit mandate, data generated will enable development of policy level implementation strategies, evaluation of determinants and important outcomes of effective implementation, and design of future mandates to improve fit and fidelity.

Safety of 3'-sialyllactose (3'-SL) sodium salt produced by a derivative strain (Escherichia coli NEO3) of E. coli W (ATCC 9637) as a Novel Food pursuant to Regulation (EU) 2015/2283.

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 sialic acid, d-glucose, d-lactose, 3'-sialyllactulose and 6'-sialyllactose sodium salts and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli NEO3) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF to a variety of foods, including infant formula and follow-on formula, food for special medical purposes and food supplements (FS). The target population is the general population. The applicant applies for the same uses and use levels as already assessed for 3'-SL sodium salt produced by a genetically modified strain of E. coli K-12 DH1, with the exception for the use in FS, which is proposed to be higher (from 0.5 to 1.0 g/day) in individuals from 3 years of age. Since the NF as a food ingredient would be consumed at the same extent as the already assessed 3'-SL sodium salt, no new estimates of the intakes have been carried out. The Panel notes that the maximum daily intake of 3'-SL from the proposed use of the NF in FS for individuals from 3 years of age (1.0 g/day) is lower than the estimated highest mean daily intake of 3'-SL in breastfed infants. FS are not intended to be used if other sources of 3'-SL are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Decoding the endometrial niche of Asherman's Syndrome at single-cell resolution.

Asherman's Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman's Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman's Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment.

Cultural Consensus Modeling to identify culturally relevant reasons for and against suicide among Black adolescents.

INTRODUCTION: The development of evidence-based treatments relies on accurate theoretical frameworks sensitive to the lived realities of the populations from which they are derived. Yet, the perspectives of Black youth are vastly underrepresented in extant theories of suicidal behavior. Cultural Consensus Modeling provides an evidence-based approach for developing a culturally informed understanding of suicide risk among Black youth. METHOD: Participants were 50 Black adolescents (Mage = 16.20 years; 76.0% male) who completed Phase 1 of a Cultural Consensus Modeling study. Participants freely listed reasons for suicide and reasons for living among similar peer Black youth. Responses were synthesized and coded for major themes. RESULTS: The most common reasons for suicide were racism (40%), depression (32%), poverty (26%), and bullying (22%). The most common reasons for living were family (58%), having a purpose or goals (36%), friends (30%), and hope for a better future (26%). CONCLUSION: Responses highlighted issues of racism and social justice, depression, and poverty, as well as the protective role of relationships, living for the future, and contributing to Black empowerment. Future research should utilize Cultural Consensus Modeling to elevate the voices of Black youth, improving extant theories of suicide, and identifying unique mechanisms or opportunities for prevention.

Accelerometer-based detection of African swine fever infection in wild boar.

Infectious wildlife diseases that circulate at the interface with domestic animals pose significant threats worldwide and require early detection and warning. Although animal tracking technologies are used to discern behavioural changes, they are rarely used to monitor wildlife diseases. Common disease-induced behavioural changes include reduced activity and lethargy ('sickness behaviour'). Here, we investigated whether accelerometer sensors could detect the onset of African swine fever (ASF), a viral infection that induces high mortality in suids for which no vaccine is currently available. Taking advantage of an experiment designed to test an oral ASF vaccine, we equipped 12 wild boars with an accelerometer tag and quantified how ASF affects their activity pattern and behavioural fingerprint, using overall dynamic body acceleration. Wild boars showed a daily reduction in activity of 10-20% from the healthy to the viremia phase. Using change point statistics and comparing healthy individuals living in semi-free and free-ranging conditions, we show how the onset of disease-induced sickness can be detected and how such early detection could work in natural settings. Timely detection of infection in animals is crucial for disease surveillance and control, and accelerometer technology on sentinel animals provides a viable complementary tool to existing disease management approaches.

Low transmission risk of African swine fever virus between wild boar infected by an attenuated isolate and susceptible domestic pigs.

African swine fever (ASF) is a lethal infectious disease that affects domestic and wild pigs. This complex virus has already affected five continents and more than 70 countries and is considered to be the main threat to the global swine industry. The disease can potentially be transmitted directly through contact with infectious animals, or indirectly by means of contaminated feed or environments. Nevertheless, the knowledge regarding the transmission patterns of different ASF virus isolates at the wildlife-livestock interface is still limited. We have, therefore, assessed the potential transmission of an attenuated ASF virus isolate between infectious wild boar and directly exposed domestic pig. We registered 3,369 interspecific interactions between animals, which were brief and mostly initiated by wild boar. The major patterns observed during the study were head-to-head contact owing to sniffing, thus suggesting a high probability of pathogen transmission. However, only one of the five domestic pigs had a short period of viremia and became serologically positive for ASF virus antibodies. It was additionally discovered that the wild boar did not transmit the virulent virus isolate to the domestic pigs, which suggests that the presence of attenuated ASF virus isolates in affected areas may control the spreading of other more virulent isolates. These outcomes may help make decisions related to large-scale targeted management actions against ASF in field conditions.

Combined Effect of Citric Acid and Polyphenol-Rich Grape Seed Extract towards Bioactive Smart Food Packaging Systems.

Alginate films (2% w·v-1) were prepared with varying concentrations (5-20% w/w) of citric acid and aqueous grape seed extract (GSE) filtrate (11.66 ± 1.32 g GAE/L) using the solvent-evaporation method. Crosslinking alginate via ester bonds (FTIR analysis) with citric acid up to 10% (w/w) led to a 33% increase in tensile strength, a 34% reduction in water vapor transmission rate (WVTR), and had no impact on elongation at break. Crosslinking alginate with citric acid in the presence of GSE increased the tensile strength by 17%, decreased WVTR by 21%, and significantly improved DPPH scavenging activity. Moreover, after incubation for 24 h at 37 °C, the film-forming solutions exhibited increased antimicrobial activity, resulting in 0.5- and 2.5-log reductions for Escherichia coli and Staphylococcus aureus, respectively, compared to the values obtained without the addition of GSE. The stronger inhibitory effect observed against Gram-positive bacteria can be attributed to the unique composition and structure of their cell walls, which creates a barrier that restricts the penetration of polyphenols into the cells. The pH adjustment of the GSE film-forming solution from 2.0 to 10.0 shifted the UV/VIS absorption spectra, resulting in a colour change from yellow to red. The findings of this study have showcased the potential of combining GSE and citric acid to enhance the functionality and bioactivity of alginate films for applications in smart food packaging.

Safety of 3-fucosyllactose (3-FL) produced by a derivative strain of Escherichia coli K-12 DH1 as a novel food pursuant to Regulation (EU) 2015/2283.

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-fucosyllactose (3-FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharide (HiMO) 3-FL, but it also contains d-lactose, l-fucose, 3-fucosyllactulose and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli K-12 DH1 MDO MAP1834) of E. coli K-12 DH1 (DSM 4235). The information provided on the manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF to a variety of foods, including infant formula and follow-on formula, food for special medical purposes and food supplements (FS). The target population is the general population. The anticipated daily intake of 3-FL from both proposed and combined (authorised and proposed) uses at their respective maximum use levels in all population categories does not exceed the highest intake level of 3-FL from human milk in infants on a body weight basis. The intake of 3-FL in breastfed infants on a body weight basis is expected to be safe also for other population groups. The intake of other carbohydrate-type compounds structurally related to 3-FL is also considered of no safety concern. FS are not intended to be used if other foods with added 3-FL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Safety of 6'-sialyllactose (6'-SL) sodium salt produced by a derivative strain (Escherichia coli NEO6) of E. coli W (ATCC 9637) as a Novel Food pursuant to Regulation (EU) 2015/2283.

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 sialic acid, d-glucose, d-lactose, 6'-sialyllactulose sodium salt, 3'-sialyllactose (3'-SL) sodium salt and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli NEO6) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF to a variety of foods, including infant formula and follow-on formula, food for special medical purposes and food supplements (FS). The target population is the general population. The applicant applies for the same uses and use levels already assessed for 6'-SL sodium salt produced by fermentation by a genetically modified strain of E. coli K-12 DH1. Therefore, since the NF would be consumed at the same extent as the already assessed 6'-SL sodium salt, no new estimates of the intake have been carried out. Similarly, FS are not intended to be used if other foods with added 6'-SL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells.

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

Functional alterations of two novel MC4R mutations found in Argentinian pediatric patients with early onset obesity.

Loss-of-function mutations in melanocortin-4 receptor (MC4R) are the most common cause of monogenic obesity, a severe type of early-onset obesity. Our aim was to determine the prevalence of MC4R mutations in a cohort of 97 Argentinian children with early-onset obesity. We found two novel mutations (p.V52E and p.G233S) and estimated a prevalence of 2.1%. We investigated the pathogenicity of mutations in HEK293T cells expressing wild-type or mutant MC4R and found that both mutants exhibited reduced plasma membrane expression and altered agonist-induced cAMP responses, with no changes in basal activity. Besides, MC4R G233S mutant demonstrated an altered agonist-dependent inhibition of voltage-gated calcium channels type 2.2. Results using a Gαs protein inhibitor suggest that the G233S mutation could be recruiting a different G-protein signaling pathway. The identification of new mutations in MC4R and characterization of their functional impact provide tools for the diagnosis and treatment of monogenic obesity.

Safety of 2'-fucosyllactose (2'-FL) produced by a derivative strain (APC199) of Corynebacterium glutamicumATCC 13032 as a novel food pursuant to Regulation (EU) 2015/2283.

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 2'-fucosyllactose (2'-FL) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharide (HiMO) 2'-FL, but it also contains d-lactose, l-fucose, 3-fucosyllactose, difucosyllactose, d-glucose and d-galactose. The NF is produced by fermentation with a genetically modified strain (APC199) of Corynebacterium glutamicum ATCC 13032. 2'-FL, when chemically synthesised or produced by fermentation with derivative strains of Escherichia coli K-12 DH1 or E. coli BL21 (DE3), is already authorised and included in the EU list of NFs. This application refers to a change in the production process and specifications, while target population, conditions of use and consequently, the anticipated intake remain unchanged. The information provided on the identity, production process, composition and specifications of the NF does not raise safety concerns. The intake of other carbohydrate-type compounds structurally related to 2'-FL is also considered of no safety concern. In line with other milk oligosaccharides that are natural components of human milk, the safety assessment of this NF is mainly based on the comparison between the intake of breastfed infants and the estimated intake as NF. Given that the NF would be consumed at the same extent as the already authorised 2'-FL, 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.

Mechanical, Barrier, Antioxidant and Antimicrobial Properties of Alginate Films: Effect of Seaweed Powder and Plasma-Activated Water.

The incorporation of natural fillers such as seaweed may potentially enhance the properties of biopolymer films. In this study, we investigated the effect of seaweed powder as a bio-filler in alginate-based films at different concentrations (10, 30, and 50%, w/w alginate) and particle sizes (100 and 200 µm) on the mechanical, barrier, antioxidant, and antimicrobial properties of alginate which are essential for food packaging applications. Initially, mechanical properties of the alginate films prepared at different temperatures were evaluated to find the optimal temperature for preparing alginate solution. The addition of seaweed powder did not have any positive effect on the mechanical properties of the alginate films. However, the barrier (water vapor transmission rate) and antioxidant properties were improved with the addition of seaweed filler regardless of concentration. In addition, selected films were prepared in plasma-activated water (PAW). The mechanical properties (tensile strength, but not elongation at break) of the films prepared with PAW improved compared to the films prepared in distilled water, while a significant decrease was observed when incorporated with the seaweed filler. The films prepared in PAW also showed improved barrier properties compared to those prepared in distilled water. The antimicrobial activity of the alginate-seaweed film-forming solution was in general more pronounced when prepared with PAW and stored at 10 °C, particularly at the highest concentration of the film-forming solution (83.3% v/v). A more pronounced inhibitory effect was observed on the Gram-positive S. aureus than on the Gram-negative E. coli, which has been attributed to the different composition and structure of the respective cell walls. This study has demonstrated the potential of seaweed filler in combination with PAW towards enhanced functionality and bioactivity of alginate films for potential food packaging applications.