Identifying cell types present in lung biopsies can provide critical information on pathological processes, tissue organization, and organ health, and are valuable in both clinical and research settings. Knowledge of cell types, their distributions, and spatial relationships can assist pathologists and researchers in diagnosis, prognosis, and mechanistic investigations. Multiplex imaging technologies such as Phenocycler™, based on co-detection by indexing (CODEX) can provide whole-slide spatial maps of protein expression and can accurately identify cell types; however, CODEX shares limitations including high costs of antibodies and reagents, as well as labor-intensive conjugation and validation steps. In contrast, hematoxylin and eosin (H&E) staining is inexpensive and routinely available, offering the potential for scalable cell-type mapping if robust prediction can be achieved directly from histology. In this work, we develop a deep learning pipeline to automatically detect cell types in H&E-stained lung tissue sections by leveraging ground-truth annotations from paired CODEX images. The dataset comprises over 2.3 million labeled cells, segmented from lung tissue sections obtained from multiple donors, and grouped into five broad classes: epithelial, immune, endothelial, stromal, and contractile. We train a DeepLabV3+ semantic segmentation model with ResNet backbone on patches from these annotated slides and reserve a subset for testing. Our model achieved 51.3% balanced accuracy across five classes, which is ~2.5x the random-guessing baseline of 20%. This framework demonstrates the feasibility of approximating multiplexed imaging-based cell type maps from routine histology.
Illuminating mechanisms at the tissue and cellular levels using indexed oligonucleotide-conjugated antibodies has further increased the resolution necessary to study pathological disease at microscopic levels. CO-Detection by indEXing (CODEX) is a high-multiplex microscopy method that uses up to 100 unique conjugated antibodies to elucidate which cell types and cellular neighborhoods are present in the imaged tissue, to determine their positions relative to each other and their functional states for physiologically-based mechanistic derivations. The first part of these procedures describes how to conjugate antibodies to their assigned oligonucleotide. Then, these conjugates are all optimized for either formalin-fixed paraffin-embedded (FFPE) or fresh-frozen (FF) tissues. In the third part, the conjugate-stained tissue section undergoes the CODEX multicycle reaction where fluorescently labeled oligonucleotides complementary to conjugates are periodically added and removed to aid in the capture of images. Lastly, the collected images are processed to set the stage for cell type and cellular neighborhood mapping on the imaged tissue for further spatial analysis work. This method has advanced basic antibody research and translational pathology workflows as well as refining new clinical treatments by providing bioinformatic support to clinicians to help patients with various diseases, especially cancer.
Reproducible quality assessment is essential for the standardization and international comparability of herbal granule products. Gastrodiae Rhizoma granules (GRG) are widely used herbal preparations, yet analytical strategies aligned with different compendial requirements remain limited. The study aimed to establish and evaluate a Deutscher Arzneimittel-Codex (DAC)-aligned analytical strategy for the quality assessment of GRG and compare it with the Chinese national standard. A multi-dimensional analytical approach integrating thin-layer chromatography (TLC), quantitative analysis of multi-components by single marker (QAMS), and high-performance liquid chromatography (HPLC) fingerprinting was developed for GRG. TLC was assessed using DAC system suitability requirements. Gastrodin was used as the internal reference for simultaneous determination of p-hydroxybenzyl alcohol and parishins E, B, C, and A by QAMS. HPLC fingerprints were established for 15 batches of GRG and evaluated by cosine similarity analysis, principal component analysis, hierarchical clustering analysis, and radar plot visualization. The TLC method achieved clear separation of gastrodin and parishin B, satisfying DAC suitability requirements. QAMS results showed no significant difference (P > 0.05) compared with the external standard method, demonstrating good accuracy and reliability. HPLC fingerprint analysis revealed generally consistent chemical profiles among the investigated batches, while also identifying inter-batch variation in several samples. Chemometric analyses further supported the overall similarity and localized heterogeneity of the investigated batches. Compared with the Chinese national standard, the DAC-aligned strategy placed greater emphasis on chromatographic suitability and multi-component characterization. This study demonstrates the feasibility of a DAC-aligned analytical strategy for the multi-dimensional quality assessment of GRG, and offers a useful reference for cross-standard comparison and for the harmonization of quality evaluation approaches of herbal granule products.
Globally, there are differences in the way oats are regulated-particularly regarding the food labeling when oats are called gluten-free, with the potential to impact people who have celiac disease or related conditions. The potential cross-contact of oats with wheat, rye, and barley during processing is a concern for people with gluten-related disorders. This study examined selected international regulations governing gluten-free foods, the regulatory treatment of oats, and the requirements for declaring oats as an ingredient on gluten-free food labels. A comparative review of key regulatory frameworks was undertaken, including the World Health Organization and Food and Agriculture Organization-Codex Alimentarius Standard, and regulations from the United Kingdom (UK), the European Union (EU), the United States, Canada, and Australia and New Zealand (ANZ). All of the listed jurisdictions, with the exception of ANZ, define gluten-free foods as foods containing less than 20 mg gluten/kg food (20 ppm) (in the United States), or less than or equal to 20 ppm. In ANZ, a gluten-free food must not contain detectable gluten or oats, which prohibits oats from being labeled as gluten-free. The United States alone classifies oats as not containing gluten. The regulations of many countries are based on the premise that oats processed with minimizing of gluten cross-contact are safe for most individuals with celiac disease, but stipulate that oats are only permitted to be labeled gluten-free if they contain <20 ppm (in the United States) or ≤20 ppm gluten (in the remaining listed jurisdictions with the exception of ANZ). The current ANZ labeling regulations are not aligned with other international regulations, and emerging clinical evidence suggests it may be timely and impactful to re-evaluate the regulatory definitions of oats, such that individuals can safely identify products suitable for their medical requirements.
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Sound is a core component of digital games, and its integration is assumed to support learning, motivation, and positive emotions. However, empirical evidence on the role of sound in educational video games remains limited, particularly in narrative-driven educational adventure games such as digital history games. In a laboratory experiment, university students (N = 111) either played an educational history video game without sound or with additional sound features (ambient audio, character voices, and narrated codex entries providing additional historical information). Post-test measures assessed factual knowledge, triggered and maintained situational interest, and academic emotions (enjoyment and boredom). Engagement with optional supplemental historical information provided through in-game codex entries was measured using behavioral log data. We analyzed differences between the two conditions while controlling for relevant pre-test variables. Participants in the add-on sound condition did not score significantly higher on the knowledge test than those in the no-sound condition. Likewise, no statistically significant differences emerged in situational interest, enjoyment, boredom, or codex engagement between conditions. Additional analyses indicated that participants' interactions with codex entries positively predicted knowledge test performance, indicating that voluntary engagement with supplemental content contributed to learning. Our findings suggest that the presence of sound alone may not enhance academic outcomes in a narrative-driven educational video game. Additionally, our findings indicate that learning outcomes depended strongly on learners' engagement with in-game codex entries. Overall, our results on the inclusion of sound highlight the importance of examining specific design features within educational history video games.
Gluten-free regulations require analytical systems that can reliably measure gluten in wheat, rye, and barley, and, in some jurisdictions, in oats across diverse food ingredients and processed foods. Examples include baked goods, fermented products such as beer, and extruded snack foods, where processing can alter gluten extractability and antibody recognition. This review examines the scientific, analytical, and regulatory factors that determine whether gluten quantification can support robust enforcement of the 20 mg gluten/kg threshold used in most jurisdictions, with a focus on method performance criteria (MPCs). The molecular complexity of gluten and the structural changes induced by baking, fermentation, and other processing steps influence extractability and epitope availability. These effects contribute to variable results across enzyme-linked immunosorbent assays that use different antibodies, extraction chemistries, and calibration standards. The widespread use of PWG gliadin (gliadin reference material) as a calibrant supports harmonized calibration for wheat-based analyses. Still, it can introduce bias because it does not reflect the gluten composition in processed or mixed-cereal foods. Improved incurred reference materials offer advantages but are not yet widely accessible. Proficiency testing data from DLA and FAPAS show that interlaboratory agreement is strongest for unprocessed matrices and decreases in baked, fat-rich, or fermented foods. These findings highlight the need for harmonized validation and for statistical criteria that define acceptable assay performance around the regulatory threshold. Advanced techniques such as liquid chromatography-tandem mass spectrometry provide detailed peptide-level information and can support confirmation in matrices where immunoassays lose sensitivity. A coordinated, performance-based framework aligned with Codex guidance would enable analytical methods to be evaluated against common criteria for recovery, precision, and detection capability. Codex alignment also introduces method dependency, as the current Type I Codex approach for gluten relies on the R5 Méndez ELISA as the defining method. Because Type I methods are empirical defining methods, results are comparable to the definition established by that method rather than necessarily being traceable to an independent reference value for gluten. The use of a single Type I method can therefore limit comparability when laboratories use assays based on other antibodies or extraction and calibration systems, particularly in processed matrices. Integrating such MPCs into regulatory practice, proficiency testing, and private certification systems would improve the comparability of gluten testing results and improve the reliability of gluten-free labeling in international markets.
The One Health approach recognizes that antimicrobial monitoring and regulations must jointly address the risks of antimicrobial resistance in human and animal health for effective strategy and policy to achieve global public health. We operationalize this principle by integrating the World Health Organization's human importance classifications with the World Organization for Animal Health's veterinary classifications to create a hierarchical framework that prioritizes human health. This framework underpins our unique "One Health Score" (OHS), to evaluate regulatory compliance of country-specific drug residue limits with international standards of Codex. Codex, the European Union, and the United States are identified as leaders of establishing maximum residue limits to which other countries defer to. Among pairs with medical classifications, 53% fall under the pairing of Highly Important Antimicrobials (H3) for humans and Veterinary Critically Important Antimicrobials (V1) for animals. The One Health Score is non-negative for at least 50% of countries at each medical importance level. However, 21% of the countries with H3V1 MRLs are laxer than Codex. Drugs with a high market demand in animal agriculture are the most commonly regulated among countries. This suggests drugs used less in food animals may not be adequately regulated currently, despite, in some cases, being highly important for human health. Country officials must balance economic and health priorities when setting or modifying drug residue limits.
Background: Interpretation of pesticide residues in fruits requires tight integration of surveillance evidence, analytical capability, regulatory context, and mitigation data. Methods: This critical integrative review synthesises analytical chemistry, cumulative risk assessment (CRA), regulatory divergence, and mitigation evidence, strengthened by quantitative monitoring summaries and auditable regulatory examples. Routine enforcement continues to rely on validated QuEChERS extraction coupled with targeted LC-MS/MS and GC-MS/MS. High-resolution mass spectrometry (HRMS) adds unique value for metabolites, transformation products (TPs), and incident response, but its routine enforcement role remains constrained by confirmation logic and harmonised validation. Results: Monitoring shows that exposure is typically multi-residue rather than single-compound; the key interpretive challenge therefore shifts toward CRA prioritisation, sensitive-subpopulation assumptions, and transparent distinction between compliance signals and toxicological inference. We provide (i) headline compliance metrics from EU and US programmes, (ii) surveillance-derived high-frequency residue patterns and co-occurrence motifs to guide CRA prioritisation, (iii) an illustrative, traceable comparison of EU/US/Codex MRL divergence for emblematic citrus residues with EU evidence extracts and US/Codex traceability records, and (iv) mitigation evidence statements standardised by study type and transformation-product reporting. Conclusions: Pesticide residues in fruits should be interpreted through a risk-based framework that distinguishes compliance findings from toxicological concern, prioritises relevant multi-residue drivers, and evaluates mitigation according to both residue reduction and transformation-product uncertainty.
Accurate cell segmentation is an essential step for quantitative analysis of biological imaging data. Recent advances in deep learning have led to the development of generalist segmentation models that perform robustly across multiple imaging modalities, including label-free phase contrast, fluorescence cell culture, and multiplexed fluorescence tissue imaging. However, systematic comparisons of these models at the level of downstream biological analysis remain limited. To address this gap, we evaluated several recent segmentation models, including Cellpose cyto3, Cellpose-SAM, μSAM, and CellSAM, on phase contrast and fluorescence cell culture images. In addition, Mesmer and InstanSeg were included for benchmarking on multiplexed fluorescence tissue images generated using CO-Detection by IndEXing (CODEX). We found that Cellpose-SAM achieved strong performance on phase contrast images, while SAM-based models consistently performed well on fluorescence cell culture data. In contrast, no single model consistently outperformed others on CODEX datasets. Instead, each model exhibited distinct strengths and limitations, which led to differences in downstream analyses, including clustering and cell type identification. Together, our study emphasizes the importance of selecting segmentation models based on dataset characteristics and analytical goals, rather than relying on a single universal approach.
Bacteriophage-based products are gaining attention as effective tools to reduce harmful germs in food and combat antimicrobial resistance throughout the food production process. However, in South America, their use is still limited because of complicated regulations and inconsistent evidence requirements. This review aims to (i) explore the current scientific and technological landscape of using bacteriophages in South American food systems, (ii) identify main regulatory challenges that impact their classification, approval, and use, and (iii) highlight the need for consistent international guidelines, especially from Codex Alimentarius, to help safely and effectively incorporate phage-based products in food. Research on phage-based products is growing, but it is not consistent across different regions. There are more patents and advancements in biotechnology, but they are limited to certain areas. Although progress is being made, the regulatory frameworks are still unclear, especially when it comes to how these products are classified, labeled, and monitored for safety. To address these gaps, risk-based guidelines are needed. These should define product categories and claims, set safety standards, and include rules for tracking products and monitoring them after they hit the market. Creating a new Codex Alimentarius project on phage-based products could help establish global guidelines that promote safe use, reduce uncertainty in regulations, and improve trade in food markets around the world.
Ampicillin residues in animal-derived foods may cause allergic reactions and promote antimicrobial resistance in consumers; however, data on residue behavior in poultry remain limited. This study aimed to evaluate the depletion of ampicillin in muscle and skin plus fat of broiler chickens. Thirty birds were treated with ampicillin intramuscularly (20 mg kg-1 every 24 h for three days) and sacrificed at 0.5, 1, 2, 5, and 9 days post-administration. Samples were analyzed by liquid chromatography coupled with tandem mass spectrometry, a method successfully validated according to Commission Implementing Regulation (EU) 2021/808, VICH GL49 and GL2. Quantification was performed by linear regression from matrix-matched calibration curves. Residue depletion was evaluated following the European Medicines Agency guidelines. Ampicillin residues in muscle were detected only during the first 24 h post-administration (6.50-8.48 µg kg-1). Residues in skin plus fat remained detectable until day 5 post-administration (6.87-59.88 µg kg-1). Based on this, the withdrawal period calculated for skin plus fat was 9 days considering EU maximum residue limit (MRL) and 19 days considering method limit of quantification, with 95% confidence. These results provide critical data on ampicillin residue kinetics under controlled experimental conditions, supporting risk assessments and the establishment of MRLs in broiler chickens by the Codex Alimentarius.
Glyphosate, a broad-spectrum herbicide extensively utilized in global agriculture, had raised significant concerns regarding its persistence in food products alongside potential health risks. Although several studies have detected glyphosate residues in various food items, data on its presence in cereal-based products frequently consumed by Lebanese children and adolescents remain limited. This study intends to assess glyphosate concentrations in breakfast cereals and wheat-based snacks available on the Lebanese market and to estimate dietary exposure among Lebanese children and adolescents. A total of 62 stock keeping units (SKUs), including breakfast cereals (n = 21) and a convenient sample of wheat-based snacks (n = 41) were retrieved from major supermarkets in Beirut and Mount Lebanon between 2022 and 2024. Enzyme-linked immunosorbent assay (ELISA) technique was used to quantify glyphosate residues. Detected glyphosate levels were compared with the maximum residue levels (MRL) of 30 mg/kg set by Codex Alimentarius and contamination patterns were compared statistically by product category, country of origin, and main ingredient. All samples (100%) had detectable glyphosate residues, yet all levels were below the established MRLs. Breakfast cereals displayed significantly higher glyphosate concentrations (median: 44.45 ppb) when compared to snacks (median: 36.38 ppb) (p = 0.003). Imported snacks had significantly higher residue levels (49.7 ppb) than locally produced items (30.6 ppb) (p = 0.0015). The dietary exposure assessment demonstrated that children's exposure to glyphosate through frequent consumption of breakfast cereals and snacks was minimal, with the highest exposure observed in the 4-8-year age group representing less than 0.031% of the most conservative Acceptable Daily Intake set by regulatory authorities. However, considering children's and adolescents' vulnerability to chronic, low-dose exposure, extensive monitoring programs targeting foods consumed by these age groups, along with additional research on total dietary exposure to glyphosate from various food sources, are recommended. These findings should be interpreted with caution given ongoing scientific debate regarding the adequacy of existing safety limits and potential long-term health effects.
Deoxynivalenol (DON) is a harmful mycotoxin that poses significant health risks to humans and animals through contaminated cereals and feedstuffs. This study describes the design and characterization of a bifunctional fusion enzyme that catalyzes the single-enzyme cascade degradation of DON into the less toxic product 3-epi-DON. Six fusion enzymes (QAF1-3 and AQF1-3) were constructed by fusing DEQDDH and AKR13B2 in reverse orientations using flexible (GGGGS)n linkers (n = 1-3). The fusion enzyme QAF2 exhibited the highest degradation efficiency, achieving a 94.99 ± 2.95% degradation rate of DON (4 μg/mL) within 12 h, with 3-epi-DON as the sole product. QAF2 displayed optimal activity at pH 6.0 and 40 °C, with a specific activity of 4.88 ± 0.5 U/mg. While its degradation efficiency was comparable to that of the free two-enzyme cascade, QAF2 demonstrated markedly superior pH stability and thermostability, maintaining more than 90% relative activity at pH 5-8 and 30-45 °C. In three practical feed matrices, QAF2 reduced DON concentrations below the regulatory limits of Codex Alimentarius (CXS 193-1995) and the European Union (EC 1881/2006) within 3 h, and achieved more than 89% degradation after 12 h. These findings establish QAF2 as a promising biocatalyst for DON detoxification in food and feed applications.
Food fraud through species substitution has been reported for edible gastropods, particularly in processed products where morphological identification is not feasible. DNA-based methods have been used to identify gastropod species in food products, but are largely limited to targeted analyses. To address this limitation, we applied, for the first time, an untargeted DNA metabarcoding approach to detect and identify gastropod species in processed foods. Mitochondrial 16S rDNA fragments of approximately 150 bp were analyzed. In 17 of 18 reference samples, taxa were resolved to genus or species level, in agreement with Codex Alimentarius Austriacus guidelines, facilitated by a curated reference database that enables assignments across freshwater, marine, and terrestrial gastropods. Thirteen DNA extract mixtures were analyzed for method validation, yielding few false-negative results for minor components. These likely resulted from primer-template mismatches causing amplification bias. Analysis of 20 commercial food products revealed seven mislabeled samples. The approach is compatible with established in-house metabarcoding assays targeting other animal species and consequently complements broad application across various taxa. This qualitative screening tool is suitable for routine food authentication, supporting supply chain transparency and regulatory monitoring.
To explore the effects of artificial sweeteners on reduced antibiotic susceptibility and expression of antibiotic resistance and virulence-related genes in bacteria exposed to permitted daily intake levels. We first evaluated the antibacterial effects of five commonly used artificial sweeteners (saccharin, cyclamate, aspartame, acesulfame potassium, and sucralose) on Escherichia coli and Bacillus subtilis, and detected changes in reactive oxygen species (ROS) production. We investigated the expression of genes associated with resistance, oxidative stress, and virulence using transcriptome sequencing after 2 h of exposure. The minimum inhibitory concentration (MIC) of artificial sweeteners for E. coli and B. subtilis were higher than the Codex Alimentarius Commission (CAC) defined daily intake limits. Except for saccharin, these sweeteners did not significantly affect the bacterial growth within 48 h. However, at these limiting concentrations, artificial sweeteners are associated with reduced antibiotic susceptibility and upregulation of resistance-related genes and enhance ROS production. Transcriptome analysis at 2 h revealed that artificial sweeteners upregulated genes associated with resistance, oxidative stress, and virulence compared to the glucose control. Additionally, we observed the effects of artificial sweeteners on iron uptake-related genes in E. coli, suggesting potential implications for bacterial ferroptosis that require further validation. Exposure to artificial sweeteners at CAC-permitted doses is associated with reduced antibiotic susceptibility and may affect bacterial function. Therefore, the safety of artificial sweeteners as substitutes for natural sugars requires careful consideration and further in vivo validation.
To summarize current advances in risk-based management of unintended food allergen presence, with a particular focus on Japan's unique 10-ppm labeling threshold and precautionary allergen labeling (PAL) prohibition, and to contextualize these within emerging international frameworks based on eliciting doses (EDs) and reference doses (RfDs). Quantitative risk assessment has shown that the aforementioned threshold offers a wide safety margin for most consumers allergic to egg, milk, and wheat, with estimated inadvertent exposures below the FAO/WHO RfDs. However, this threshold is associated with overly stringent requirements and a substantial management burden for some allergens, such as crustaceans. The FAO/WHO guidelines, Voluntary Incidental Trace Allergen Labeling (VITAL) 4.0, Codex, and regional initiatives are converging on ED-based RfDs alongside HACCP-based allergen management. In Japan, rapid shifts in nut consumption have altered allergy prevalence and ED distributions, highlighting the need for periodic threshold updates. Future allergen policy should integrate robust thresholds with realistic, HACCP-based allergen management in packaged foods and food-service settings, anticipate shifting dietary patterns, and support internationally harmonized, evidence-based PAL that enhances the safety and quality of life of food-allergic individuals.
People living with HIV (PWH) with interrupted medical care have a higher risk of viral rebound. We investigated the factors associated with loss to follow-up (LTFU) by combining socioeconomic data from three research cohorts with the nationwide follow-up data of the French Hospital Database on HIV (ANRS-CO4-FHDH). We included individuals who entered the ANRS PRIMO (primary HIV-1 infection), CODEX (HIV-1 controllers) and COPANA (recent HIV-1 diagnosis) cohorts. Data were then linked to those of the FHDH. LTFU was defined as no clinical follow-up for ≥ 24 months on December 31, 2021. Fine and Gray's subdistribution hazard models taking the competing risk of death into account were used to identify factors associated with LTFU. We included 2935 PWH in this study: 576 women, 1879 MSM (men who have sex with men) and 480 MSW (men who have sex with women). In total, 605 participants were lost to follow-up in terms of hospital care. The cumulative incidence of LTFU was 16% (95% confidence interval (CI): 15-18) at 10 years. Migrant MSM and MSW had a higher risk of LTFU than French-born MSM (adjusted subdistribution hazard ratios (aSHR): 1.68 [95%CI: 1.27-2.21] and 1.38 [0.98-1.96], respectively). Younger age, unemployment (aSHR: 1.24 [1.01-1.53]), antidepressant prescription (aSHR: 1.80 [1.21-2.67]) at inclusion, and lack of ART initiation (aSHR: 8.33 [6.25-12.50]) during follow-up were also associated with a higher risk of LTFU. A migrant background, unemployment and antidepressant prescription were associated with LTFU, highlighting the need for targeted support for vulnerable groups to improve engagement with care.
Table olive processing comprises multiple stages in which physical, chemical, and biological hazards may occur. Although risk assessment is a core element of Hazard Analysis and Critical Control Points (HACCP) systems, the selection of assessment tools remains insufficiently standardized. This study compared a 4 × 4 risk matrix and Failure Mode and Effects Analysis (FMEA) for hazard evaluation in Spanish-style and Californian-style table olive processing. Hazards were assessed across 41 processing stages for Spanish-style olives and selected key stages for Californian-style olives using probability × severity in the 4 × 4 matrix and severity × occurrence × detection in FMEA. Significant hazards were further evaluated using the Codex Alimentarius decision tree to identify critical control points (CCPs) and strengthened prerequisite programs (PRPs). Both tools identified similar significant hazards, including biological hazards associated with fermentation, brine management, storage, container sealing, and heat treatment, as well as physical hazards from foreign bodies and chemical hazards related to heavy metals, pesticide residues, mycotoxins, and food-contact material migration. FMEA provided greater analytical detail through the detection parameter, whereas the 4 × 4 matrix was simpler and more practical for complex flow diagrams. Overall, both tools were suitable for HACCP-based risk assessment in table olive processing.