Biofilm formation in food industries: Challenges and control strategies for food safety.

Various pathogens have the ability to grow on food matrices and instruments. This grow may reach to form biofilms. Bacterial biofilms are community of microorganisms embedded in extracellular polymeric substances (EPSs) containing lipids, DNA, proteins, and polysaccharides. These EPSs provide a tolerance and favorable living condition for microorganisms. Biofilm formations could not only contribute a risk for food safety but also have negative impacts on healthcare sector. Once biofilms form, they reveal resistances to traditional detergents and disinfectants, leading to cross-contamination. Inhibition of biofilms formation and abolition of mature biofilms is the main target for controlling of biofilm hazards in the food industry. Some novel eco-friendly technologies such as ultrasound, ultraviolet, cold plasma, magnetic nanoparticles, different chemicals additives as vitamins, D-amino acids, enzymes, antimicrobial peptides, and many other inhibitors provide a significant value on biofilm inhibition. These anti-biofilm agents represent promising tools for food industries and researchers to interfere with different phases of biofilms including adherence, quorum sensing molecules, and cell-to-cell communication. This perspective review highlights the biofilm formation mechanisms, issues associated with biofilms, environmental factors influencing bacterial biofilm development, and recent strategies employed to control biofilm-forming bacteria in the food industry. Further studies are still needed to explore the effects of biofilm regulation in food industries and exploit more regulation strategies for improving the quality and decreasing economic losses.

Evaluation of in silico model predictions for mammalian acute oral toxicity and regulatory application in pesticide hazard and risk assessment.

The United States Environmental Protection Agency (USEPA) uses the lethal dose 50% (LD50) value from in vivo rat acute oral toxicity studies for pesticide product label precautionary statements and environmental risk assessment (RA). The Collaborative Acute Toxicity Modeling Suite (CATMoS) is a quantitative structure-activity relationship (QSAR)-based in silico approach to predict rat acute oral toxicity that has the potential to reduce animal use when registering a new pesticide technical grade active ingredient (TGAI). This analysis compared LD50 values predicted by CATMoS to empirical values from in vivo studies for the TGAIs of 177 conventional pesticides. The accuracy and reliability of the model predictions were assessed relative to the empirical data in terms of USEPA acute oral toxicity categories and discrete LD50 values for each chemical. CATMoS was most reliable at placing pesticide TGAIs in acute toxicity categories III (>500-5000 mg/kg) and IV (>5000 mg/kg), with 88% categorical concordance for 165 chemicals with empirical in vivo LD50 values ≥ 500 mg/kg. When considering an LD50 for RA, CATMoS predictions of 2000 mg/kg and higher were found to agree with empirical values from limit tests (i.e., single, high-dose tests) or definitive results over 2000 mg/kg with few exceptions.

Alternatives of Animal Models for Biomedical Research: a Comprehensive Review of Modern Approaches.

Biomedical research has long relied on animal models to unravel the intricacies of human physiology and pathology. However, concerns surrounding ethics, expenses, and inherent species differences have catalyzed the exploration of alternative avenues. The contemporary alternatives to traditional animal models in biomedical research delve into three main categories of alternative approaches: in vitro models, in vertebrate models, and in silico models. This unique approach to artificial intelligence and machine learning has been a keen interest to be used in different biomedical research. The main goal of this review is to serve as a guide to researchers seeking novel avenues for their investigations and underscores the importance of considering alternative models in the pursuit of scientific knowledge and medical breakthroughs, including showcasing the broad spectrum of modern approaches that are revolutionizing biomedical research and leading the way toward a more ethical, efficient, and innovative future. Models can insight into cellular processes, developmental biology, drug interaction, assessing toxicology, and understanding molecular mechanisms.

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology.

A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.

Strategies for Traceability to Prevent Unauthorised GMOs (Including NGTs) in the EU: State of the Art and Possible Alternative Approaches.

The EU's regulatory framework for genetically modified organisms (GMOs) was developed for "classical" transgenic GMOs, yet advancements in so-called "new genomic techniques (NGTs)" have led to implementation challenges regarding detection and identification. As traceability can complement detection and identification strategies, improvements to the existing traceability strategy for GMOs are investigated in this study. Our results are based on a comprehensive analysis of existing traceability systems for globally traded agricultural products, with a focus on soy. Alternative traceability strategies in other sectors were also analysed. One focus was on traceability strategies for products with characteristics for which there are no analytical verification methods. Examples include imports of "conflict minerals" into the EU. The so-called EU Conflict Minerals Regulation requires importers of certain raw materials to carry out due diligence in the supply chain. Due diligence regulations, such as the EU's Conflict Minerals Regulation, can legally oblige companies to take responsibility for certain risks in their supply chains. They can also require the importer to prove the regional origin of imported goods. The insights from those alternative traceability systems are transferred to products that might contain GMOs. When applied to the issue of GMOs, we propose reversing the burden of proof: All companies importing agricultural commodities must endeavour to identify risks of unauthorised GMOs (including NGTs) in their supply chain and, where appropriate, take measures to minimise the risk to raw material imports. The publication concludes that traceability is a means to an end and serves as a prerequisite for due diligence in order to minimise the risk of GMO contamination in supply chains. The exemplary transfer of due diligence to a company in the food industry illustrates the potential benefits of mandatory due diligence, particularly for stakeholders actively managing non-GMO supply chains.

Bioluminescent Models to Evaluate the Efficiency of Light-Based Antibacterial Approaches.

The emergence of microbial resistance to antimicrobials among several common pathogenic microbial strains is an increasing problem worldwide. Thus, it is urgent to develop not only new antimicrobial therapeutics to fight microbial infections, but also new effective, rapid, and inexpensive methods to monitor the efficacy of these new therapeutics. Antimicrobial photodynamic therapy (aPDT) and antimicrobial blue light (aBL) therapy are receiving considerable attention for their antimicrobial potential and represent realistic alternatives to antibiotics. To monitor the photoinactivation process provided by aPDT and aBL, faster and more effective methods are required instead of laborious conventional plating and overnight incubation procedures. Bioluminescent microbial models are very interesting in this context. Light emission from bioluminescent microorganisms is a highly sensitive indication of their metabolic activity and can be used to monitor, in real time, the effects of antimicrobial agents and therapeutics. This chapter reviews the efforts of the scientific community concerning the development of in vitro, ex vivo, and in vivo bioluminescent bacterial models and their potential to evaluate the efficiency of aPDT and aBL in the inactivation of bacteria.

Factors Affecting Dental Students' Comfort with Online Synchronous Learning.

Background: The COVID-19 pandemic caused many universities to expand their use of videoconferencing technology to continue academic coursework. This study examines dental students' experience, comfort levels, and preferences with videoconferencing. Methods: Of 100 s-year US dental students enrolled in a local anesthesia course, 54 completed a survey following an online synchronous lecture given in August 2020. Survey questions asked about prior experience with videoconferencing, comfort levels with online and traditional classes, and reasons for not turning on their video (showing their face). Results: Overall, 48.2% had little or no experience with videoconferencing prior to March 2020. Students were more comfortable with in-classroom parameters (listening, asking questions, answering questions, and interacting in small groups (breakouts)) than with online synchronous learning, although differences were not significant (p's > 0.10). Regression analyses showed there were significant positive associations between videoconferencing experience and comfort with both answering questions and interacting in breakouts (B = 0.55, p = 0.04 and B = 0.54, p = 0.03, respectively). Students reported being more comfortable during in-classroom breakouts than in breakouts using videoconferencing (p = 0.003). Main reasons for students not turning on their cameras were that they did not want to dress up (48.1%), other students were not using their video features (46.3%), and they felt they did not look good (35.5%). Conclusions: Dental students were somewhat more comfortable with traditional in-person vs. online classroom parameters. Prior experience with videoconferencing was associated with increased comfort with synchronous learning, suggesting that after the pandemic, it may be beneficial to structure dental school curricula as a hybrid learning experience with both in-person and online synchronous courses.

Systematic Review on Endovascular Access to Intracranial Arteries for Mechanical Thrombectomy in Acute Ischemic Stroke.

PURPOSE: In acute ischemic stroke for large vessel occlusions, delayed or failed access to intracranial occlusions has a negative impact on procedural and clinical outcomes. The aim of this review is to identify and quantify access failures and challenges in mechanical thrombectomy. METHODS: A systematic literature review of PubMed and Scopus databases from January 2014 to October 2020 was performed. Articles reporting consecutive patients were used to calculate a crude failure rate of femoral and alternative accesses. RESULTS: A total of 50 articles met the inclusion criteria, totalling 12,838 interventions. Failure to access the occlusion through transfemoral access occurred in 4.4% of patients, most commonly due to challenging supra-aortic vessel anatomy, decreasing to 3.6% when all alternative access routes were attempted. Failed access from alternative routes (direct carotid, radial and brachial approaches) attempted first-line or after failed femoral attempt were reported in 7.3% of patients. The occurrence rate of potentially challenging features (anatomical, diseases or others) ranged from 4.7% to 47.4%, primarily impacting the access time, procedure time, recanalization and clinical outcomes. CONCLUSION: Failure to access the occlusion is a significant contributor to failed recanalization, regardless of access routes. Challenging, but eventually successful access is also a relevant factor in procedural and clinical outcomes; however challenging access requires a universal definition to enable quantification, so that methods for procedural optimization can be critically assessed.

Read-across approaches: current applications and regulatory acceptance in Korea, Japan, and China.

The aim of this paper was to investigate the current status of read-across approaches in the Republic of Korea, Japan, and China in terms of applications and regulatory acceptance. In the Republic of Korea, over the last 6 years, approximately 8% of safety data records used for chemical registrations were based upon read-across, and a guideline published on the use of read-across results in 2017. In Japan, read-across is generally accepted for screening hazard classification of toxicological endpoints according to the Chemical Substances Control Law (CSCL). In China, read-across data, along with data from other animal alternatives are accepted as a data source for chemical registrations, but could be only considered when testing is not technically feasible. At present, read-across is not widely used for chemical registrations and regulatory acceptance of read-across may differ among countries in Asia. With consideration of the advantages and limitations of read-across, it is expected that read-across may soon gradually be employed in Asian countries. Thus, regulatory agencies need to prepare for this progression.

Person-Centered Care in Nursing Homes: Potential of Complementary and Alternative Approaches and Their Challenges.

A person-centered approach to care shifts the mind-set of care partners from a traditional medical model to a social model in managing chronic conditions. Using a qualitative descriptive approach, this study examines the barriers and facilitators to the implementation of person-centered care (PCC) and how the integration of complementary and alternative approaches (CAA) has the potential to improve residents' quality of life in nursing homes (NHs). Findings indicate that NHs offer a wide range of engagement activities, but these activities are not purposefully integrated into a person-centered plan of care. Factors such as turnover, "working short," supervisor support, and rising resident care needs make it challenging to implement PCC in NHs. This knowledge of the landscape of activities will help us identify and improve strategies for supporting residents at a deeper, more meaningful level. CAA has the potential to be therapeutic for residents if integrated into collaborative approaches to care.

Performance of the GHS Mixtures Equation for Predicting Acute Oral Toxicity.

Acute oral toxicity classifications are based on the estimated chemical dose causing lethality in 50 % of laboratory animals tested (LD50). Given the large number of pesticide registration applications that require acute toxicity data, an alternative to the in vivo test could greatly reduce animal testing. The United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Mixtures Equation estimates the acute toxicity of mixtures using the toxicities of mixture components. The goal of this study was to evaluate the concordance of LD50s predicted using the GHS Mixtures Equation and LD50s from the in vivo test results. Using the EPA classification system, concordance was 55 % for the full dataset (N = 671), 52 % for agrochemical formulations (N = 620), and 84 % for antimicrobial cleaning products (N = 51). Most discordant results were from substances LD50 > 2000 mg/kg (limit test) or 2000 < LD50 < 5000 mg/kg that were predicted as LD50 > 5000 mg/kg. A supplementary analysis combining all formulations with an LD50 > 500 mg/kg produced a concordance of 82 %. The lack of more toxic formulations in this dataset prevented a thorough evaluation of the GHS equation for such substances. Accordingly, our results suggest the GHS equation is helpful to predict the toxicity of mixtures, particularly those with lower toxicity.

Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 2: Optimisation of cell line assays.

During the production of clostridial vaccines large numbers of mice are used for various in-process control tests. Replacement in vitro assays had been developed for the testing of the toxins and toxoids of several clostridial species, but none of these assays had been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), a project on clostridial vaccines for veterinary use was started as part of the EDQM-co-ordinated Biological Standardisation Programme (BSP). Within the framework of this project (coded BSP130) a collaborative study was organised to evaluate Vero cell-based alternative methods to the current mouse tests used to measure: i) the toxicity of Clostridium septicum toxin, ii) the absence of toxicity of C. septicum toxoid and iii) the antigenicity of C. septicum toxoid. The principal aims of the study were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the in vitro and current in vivo tests. The study results demonstrated good concordance, but the information gathered through the study (later on called Part 1) and the participants' workshop prompted the extension of the project in order to further optimise the in vitro protocols and improve their repeatability and reproducibility, which were comparable to but not better than those of the in vivo assays in Part 1. The 3 in vitro assays to be optimised in the extension of the BSP130 project were : i) the in vitro toxin neutralisation equivalence plus (TNE+), as a replacement for the in vivo minimum lethal dose (MLD) test for quantification of the toxicity of toxin; ii) the in vitro MLD, as a replacement for the in vivo MLD test for detection of residual toxicity associated with toxoid; iii) the in vitro total combining power (TCP), as a replacement for the in vivo TCP test for quantification of the antigenicity of toxoid. At this point, the Analytical Method Transfer Laboratory of Ceva-Phylaxia (Hungary), supported by the project management team, developed suitable SOPs for the 3 in vitro assays. These optimised methods were further assessed in BSP130 through a second international collaborative study (Part 2) aimed at defining repeatability and reproducibility in different laboratories and determining the levels of improvement compared with the original in vivo tests and the initial in vitro assays used in Part 1 of the project. Fourteen laboratories, comprising 4 public sector and 10 manufacturers' medicines control laboratories, from 11 countries participated in the collaborative Part 2 study, each testing 6 different C. septicum toxins and 6 C. septicum toxoids. Improved repeatability and reproducibility were observed for the optimised assays. The results of this study confirm the suitability of these assays for in-process control of C. septicum vaccines, with better repeatability and reproducibility than their in vivo equivalents. It is expected that, with appropriate minor changes and the use of relevant reagents, these optimised in vitro assays could be used not only for the assessment of C. septicum toxins and toxoids but for all cytotoxin-based clostridial antigens. The development and implementation of such in vitro assays would offer a great opportunity to significantly reduce animal usage, shorten the duration of QC test procedures and increase the precision of toxicity and antigenicity assays in clostridial veterinary vaccine in-process control. This would also provide more accurate and reproducible dosing of antigens in the final vaccine products, help to promote compendial acceptance and to proffer a basis for improved international harmonisation across this area of product testing.

Opportunities and Challenges for Integrating New In Vitro Methodologies in Hazard Testing and Risk Assessment.

Nanomaterials are defined as materials with at least one dimension of 100 nm or less. Their small size confers unique properties that may alter the toxicity profile when compared to larger forms of the same material, requiring additional considerations for safety assessment. There has been a rise in the development of nanomaterials for many applications, and although traditional approaches for toxicity testing may address some of the new toxicity concerns, many may not be directly applicable to nanomaterials and new tools or approaches may need to be developed. Since nanomaterials can exist in many different forms, each of which may cause different adverse biological effects, reliance on traditional in vivo models for safety assessment will simply not be feasible or sustainable, given the volume of materials that may need to be tested. It is essential to consider and develop new in vitro methods that can be applied for hazard identification and risk assessment. Many challenges are associated with using alternative approaches to ensure they are as robust and reliable as traditional in vivo approaches, but by overcoming these issues and adopting new testing strategies there are opportunities to improve safety assessments and reduce the reliance on animal-based toxicity testing strategies.

A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals.

Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.

Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 1.

Large numbers of mice are used in testing during the production of Clostridial vaccines. Previous work has indicated that cell line assays could replace mouse tests for certain aspects of this testing. Replacement assays have been developed for the testing of the toxins and toxoids of several clostridial species but none of these assays have been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), collaborative study BSP130 was initiated to evaluate Vero cell based alternative methods to the current mouse tests used to measure the toxicity of Clostridium septicum toxin (the minimum lethal dose (MLD) test), the freedom from toxicity of C. septicum toxoid (the MLD test) and the antigenicity of C. septicum toxoid (the total combining power (TCP) test). The principal aims of BSP130 were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the proposed in vitro and current in vivo TCP and MLD tests. 11 laboratories from 7 countries participated in the collaborative study and each tested 6 toxins and 6 toxoids. The participants' Vero cell lines were up to 1 000 times more sensitive than the mouse strains. The MLD assay in mice and on Vero cells generally ranked the toxins in a similar order in most of the laboratories. The TCP assay in mice and on Vero cells also generally ranked the toxoids in a similar order in most of the laboratories. The results demonstrate that the repeatability and reproducibility of the in vitro Vero cell based assays are no worse than that of the in vivo assays and that they are easily transferable to other laboratories. The concordance correlations between the in vivo and in vitro methods were for the MLD assays ρc=0.961 (log-transformed values) and ρc=0.921 (non-log-transformed values) and for the TCP assays ρc=0.968 (log-transformed values) and ρc=0.980 (non log-transformed values). These correlations are excellent showing that the Vero cell assays can be used as alternatives to the mouse tests for the assessment of C. septicum toxin MLD and toxoid TCP values. This study can be used by vaccine manufacturing companies as a guide for applying the same approach to other clostridial toxins and toxoids.

Nonanimal toxicology testing approaches for traditional and deemed tobacco products in a complex regulatory environment: Limitations, possibilities, and future directions.

The evaluation of tobacco products is complex due to a multitude of factors including product diversity, limited testing standards, and variability in user behavior. Alternative approaches in current testing paradigms have limitations that generally truncate their applicability beyond screening for hazard identification; this is also true for toxicological evaluations of tobacco products. In a regulatory context, results from tobacco product toxicity assessments are extrapolated to the in vivo condition to assess human health relevance at the individual and population level. A key limitation of alternative approaches is the difficulty and uncertainty in extrapolating results to adverse outcomes relevant to chronic tobacco exposures in humans. This difficulty and uncertainty are increased when comparing toxicological outcomes between tobacco products. Given that the interpretation and quantification of differences in assay results (e.g., mutagenicity) for tobacco product comparison may be inconclusive, the predictive value of these approaches for human risk of relevant downstream pathologies (e.g., carcinogenesis) can be limited. Development and validation of fit-for-purpose alternative approaches that are predictive of human toxicity and dose response assays with adequate sensitivity and specificity for product comparisons would help advance the field of predictive toxicology.

Assaying epigenome functions of PRMTs and their substrates.

Among the widespread and increasing number of identified post-translational modifications (PTMs), arginine methylation is catalyzed by the protein arginine methyltransferases (PRMTs) and regulates fundamental processes in cells, such as gene regulation, RNA processing, translation, and signal transduction. As epigenetic regulators, PRMTs play key roles in pluripotency, differentiation, proliferation, survival, and apoptosis, which are essential biological programs leading to development, adult homeostasis but also pathological conditions including cancer. A full understanding of the molecular mechanisms that underlie PRMT-mediated gene regulation requires the genome wide mapping of each player, i.e., PRMTs, their substrates and epigenetic marks, methyl-marks readers as well as interaction partners, in a thorough and unambiguous manner. However, despite the tremendous advances in high throughput sequencing technologies and the numerous efforts from the scientific community, the epigenomic profiling of PRMTs as well as their histone and non-histone substrates still remains a big challenge owing to obvious limitations in tools and methodologies. This review will summarize the present knowledge about the genome wide mapping of PRMTs and their substrates as well as the technical approaches currently in use. The limitations and pitfalls of the technical tools along with conventional approaches will be then discussed in detail. Finally, potential new strategies for chromatin profiling of PRMTs and histone substrates will be proposed and described.

Skin sensitization testing needs and data uses by US regulatory and research agencies.

United States regulatory and research agencies may rely upon skin sensitization test data to assess the sensitization hazards associated with dermal exposure to chemicals and products. These data are evaluated to ensure that such substances will not cause unreasonable adverse effects to human health when used appropriately. The US Consumer Product Safety Commission, the US Environmental Protection Agency, the US Food and Drug Administration, the Occupational Safety and Health Administration, the National Institute for Occupational Safety and Health, and the US Department of Defense are member agencies of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). ICCVAM seeks to identify opportunities for the use of non-animal replacements to satisfy these testing needs and requirements. This review identifies the standards, test guidelines, or guidance documents that are applicable to satisfy each of these agency's needs; the current use of animal testing and flexibility for using alternative methodologies; information needed from alternative tests to fulfill the needs for skin sensitization data; and whether data from non-animal alternative approaches are accepted by these US federal agencies.