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Estimating the impact of pesticide residue bioaccessibility in fruits on dietary exposure is a complex task in human health risk assessment. This research investigated the bioaccessibility of ten commonly used and detected pesticides in bananas and mangoes, as well as the factors influencing it, using an in vitro model. The highest bioaccessibility was observed at pH levels of 2.5 and 6.5 in the gastric and intestinal stages, respectively. Bioaccessibility decreased significantly with increasing solid/liquid ratios for most pesticides. The consumption of protein and four dietary components (carbohydrates, protein, lipids, and dietary fiber) could significantly reduce pesticide bioaccessibility by 9.89–48.32% (p < 0.05). Bioaccessibility in oral and gastric stages among four populations followed the order of adults/the elderly > children > infants, due to decreasing concentrations of α-amylase and pepsin. Pesticides in bananas generally exhibited a higher bioaccessibility (18.65–82.97%) compared to that in mangoes (11.68–87.57%). Bioaccessibility showed a negative correlation with the Log P values of the target pesticide, while no clear relationship was found between bioaccessibility and initial pesticide concentrations. Incorporating bioaccessible pesticide concentrations into risk assessments could lower dietary risk estimates by 11.85–79.57%. Assessing human exposure to pesticides based on bioaccessibility would greatly improve the accuracy of the risk assessment. [ABSTRACT FROM AUTHOR]

Given the renewed interest in boron neutron capture therapy (BNCT) and the intensified search for improved boron carriers, as well as the difficulties of coherently comparing the carriers described so far, it seems necessary to define a basic set of assays and standardized methods to be used in the early stages of boron carrier development in vitro. The selection of assays and corresponding methods is based on the practical experience of the authors and is certainly not exhaustive, but open to discussion. The proposed tests/characteristics: Solubility, lipophilicity, stability, cytotoxicity, and cellular uptake apply to both low molecular weight (up to 500 Da) and high molecular weight (5000 Da and more) boron carriers. However, the specific methods have been selected primarily for low molecular weight boron carriers; in the case of high molecular weight compounds, some of the methods may need to be adapted. [ABSTRACT FROM AUTHOR]

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment. [ABSTRACT FROM AUTHOR]

Glycine max (L.) Merr. (soybean) is a multi-purpose crop used for both animal and human feed. It is an economically and industrially important crop. It possesses many therapeutical and nutraceutical compounds. Therefore, soybean is referred to as 'Gold from the soil'. Conventional breeding approaches are laborious and time-consuming. Thus, alternative biotechnological methods, such as in vitro micropropagation, regeneration, and transformation, could be advantageous. Despite the efforts made in the field of soybean micropropagation for haploidy and doubled haploidy, especially androgenesis, the success rate accounts for only approximately 2%. Androgenesis in soybean is lacking primarily due to its recalcitrant nature and differences in the development of microspores within a flower. Haploids and doubled haploids (DHs) have contributed immensely to crop improvement programs. In this review, soybean androgenesis history, production of haploids, and doubled haploids have been highlighted. The factors responsible for the androgenic responses have also been discussed. Furthermore, the review will be helpful in understanding the challenges in the standardization of protocol for the production of haploids and DHs in soybean, which will eventually assist breeding and crop improvement. [ABSTRACT FROM AUTHOR]

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Estimating the impact of pesticide residue bioaccessibility in fruits on dietary exposure is a complex task in human health risk assessment. This research investigated the bioaccessibility of ten commonly used and detected pesticides in bananas and mangoes, as well as the factors influencing it, using an in vitro model. The highest bioaccessibility was observed at pH levels of 2.5 and 6.5 in the gastric and intestinal stages, respectively. Bioaccessibility decreased significantly with increasing solid/liquid ratios for most pesticides. The consumption of protein and four dietary components (carbohydrates, protein, lipids, and dietary fiber) could significantly reduce pesticide bioaccessibility by 9.89–48.32% (p < 0.05). Bioaccessibility in oral and gastric stages among four populations followed the order of adults/the elderly > children > infants, due to decreasing concentrations of α-amylase and pepsin. Pesticides in bananas generally exhibited a higher bioaccessibility (18.65–82.97%) compared to that in mangoes (11.68–87.57%). Bioaccessibility showed a negative correlation with the Log P values of the target pesticide, while no clear relationship was found between bioaccessibility and initial pesticide concentrations. Incorporating bioaccessible pesticide concentrations into risk assessments could lower dietary risk estimates by 11.85–79.57%. Assessing human exposure to pesticides based on bioaccessibility would greatly improve the accuracy of the risk assessment.

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study—the IN vitro Systems to PredIct REspiratory toxicity Initiative—2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans. [ABSTRACT FROM AUTHOR]

Given the renewed interest in boron neutron capture therapy (BNCT) and the intensified search for improved boron carriers, as well as the difficulties of coherently comparing the carriers described so far, it seems necessary to define a basic set of assays and standardized methods to be used in the early stages of boron carrier development in vitro. The selection of assays and corresponding methods is based on the practical experience of the authors and is certainly not exhaustive, but open to discussion. The proposed tests/characteristics: Solubility, lipophilicity, stability, cytotoxicity, and cellular uptake apply to both low molecular weight (up to 500 Da) and high molecular weight (5000 Da and more) boron carriers. However, the specific methods have been selected primarily for low molecular weight boron carriers; in the case of high molecular weight compounds, some of the methods may need to be adapted.

Bioavailability assessment in the development phase of a drug product is vital to reveal the disadvantageous properties of the substance and the possible technological interventions. However, in vivo pharmacokinetic studies provide strong evidence for drug approval applications. Human and animal studies must be designed on the basis of preliminary biorelevant experiments in vitro and ex vivo. In this article, the authors have reviewed the recent methods and techniques from the last decade that are in use for assessing the bioavailability of drug molecules and the effects of technological modifications and drug delivery systems. Four main administration routes were selected: oral, transdermal, ocular, and nasal or inhalation. Three levels of methodologies were screened for each category: in vitro techniques with artificial membranes; cell culture, including monocultures and co-cultures; and finally, experiments where tissue or organ samples were used. Reproducibility, predictability, and level of acceptance by the regulatory organizations are summarized for the readers. [ABSTRACT FROM AUTHOR]

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation. [ABSTRACT FROM AUTHOR]

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing. [ABSTRACT FROM AUTHOR]

Aluminum chlorohydrate (ACH) is a major aerosol component frequently used as the active ingredient in antiperspirants, and in vivo studies have raised a concern about its inhalation toxicity. Still, few studies have addressed its effects on the human respiratory tract. Therefore, we developed a study on ACH inhalation toxicity using an in vitro human alveolar cell model (A549 cells) with molecular and cellular markers of oxidative stress, immunotoxicity, and epigenetic changes. The chemical characterization of ACH suspensions indicated particle instability and aggregation; however, side‐scatter analysis demonstrated significant particle uptake in cells exposed to ACH. Exposure of A549 cells to non‐cytotoxic concentrations of ACH (0.25, 0.5, and 1 mg/ml) showed that ACH induced reactive oxygen species. Moreover, ACH upregulated TNF, IL6, IL8, and IL1A genes, but not the lncRNAs NEAT1 and MALAT1. Finally, no alterations on the global DNA methylation pattern (5‐methylcytosine and 5‐hydroxymethylcytosine) or the phosphorylation of histone H2AX (γ‐H2AX) were observed. Our data suggest that ACH may induce oxidative stress and inflammation on alveolar cells, and A549 cells may be useful to identify cellular and molecular events that may be associated with adverse effects on the lungs. Still, further research is needed to ensure the inhalation safety of ACH. Aluminum chlorohydrate (ACH) is an aerosol component used in antiperspirants, but its effects in the human respiratory tract are unclear. A549 cells were used as an in vitro cell model to investigate ACH lung toxicity. Despite instability and aggregation, ACH particle uptake was observed in A549 cells. Our results shows that ACH can increase reactive oxygen species and TNF, IL6, IL8, and IL1A mRNA in A549 cells. Our data may direct future research on the potential inhalation toxicity of ACH. [ABSTRACT FROM AUTHOR]

The Abnormal Toxicity Test (ATT) is an in vivo assay traditionally used in the autogenous vaccine release. That test, commonly applied to verify vaccine quality, is subject to ethical and practical limitations and should be removed as reference methods. This approach is necessary to update the regulatory framework and to apply the 3Rs principle in the pharmaceutical field. In this study, two in vitro cell viability assays were used to evaluate the potential toxicity of autogenous vaccines. Cells were treated with base-2 dilutions of autogenous vaccines, and viability was detected by MTT and ATP assays. Differences and similarities between these tests were analysed. The ATP test showed good cell viability at 1:32 or 1:64 vaccine dilutions. The MTT assay gave discordant results and vaccines were not cytotoxic from the first dilutions. For this reason, a preference has been highlighted for the ATP method, a more sensitive test, capable of providing more accurate results., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson's disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y. [ABSTRACT FROM AUTHOR]

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a large class of industrial chemicals with a ubiquitous and persistent presence in the environment. Of the thousands of PFAS used by consumers and industry, very few have been thoroughly characterized for potential adverse effects. This is especially true for the novel short‐chain (C < 8) alternatives that replaced legacy PFAS. Perfluoroalkyl and polyfluoroalkyl substances have revealed inconsistencies in the toxicokinetics predicted by animal models and empirical findings in humans. To adequately assess the possible health effects of short‐chain PFAS, there is a need for robust aggregated data sets on the mechanistic underpinnings and physiochemical properties of these alternatives. Acquiring relevant data on the health effects of short‐chain PFAS can be achieved through high‐throughput methods supported by in vitro human cell‐based models. This review briefly summarizes some of the toxicity data obtained using human cells in vitro, discusses the advantages and limitations of cell‐based models, and provides insights on potential solutions to challenges presented with the use of these methods for use in safety assessments. Studies evaluating short‐chain perfluoroalkyl and polyfluoroalkyl substances (PFAS) have revealed inconsistencies in the toxicokinetics predicted by animal models and empirical findings in humans. Acquiring relevant data on the health effects of short‐chain PFAS in humans can be achieved through high‐throughput methods supported by human cell‐based models. This review summarizes some of the short‐chain PFAS toxicity data obtained using human cells in vitro, discusses their advantages and limitations, and offers potential solutions for implementation in safety assessments. [ABSTRACT FROM AUTHOR]

The U.S. Department of Agriculture (USDA) regulates the potency testing of leptospirosis vaccines, which are administered to animals to protect against infection by Leptospira bacteria. Despite the long-term availability of in vitro test methods for assessing batch potency, the use of hamsters in lethal in vivo batch potency testing persists to varying degrees across leptospirosis vaccine manufacturers. For all manufacturers of these products, data collected from public USDA records show an estimated 40% decline in the annual use of hamsters from 2014 to 2020, with an estimated 55% decrease in the number of hamsters expected to have been used in leptospirosis vaccine potency tests (i.e., those in USDA Category E). An estimated 49,000 hamsters were used in 2020, with about 15,000 hamsters in Category E specifically. Based on this assessment, additional efforts are needed to fully implement in vitro batch potency testing as a replacement for the in vivo batch potency test. We propose steps that can be taken collaboratively by the USDA Center for Veterinary Biologics (CVB), manufacturers of leptospirosis vaccines, government agencies, and non-governmental organizations to accelerate broader use of the in vitro approach. [ABSTRACT FROM AUTHOR]

Current highly sensitive methods for rabies virus and rabies antibodies detection in biological material can be used not only for diagnosis and experimental research, but also for the production of antirabies medicines used for postexposure prophylaxis. The aim of the study was to analyse existing methods for rabies virus and rabies antibodies detection and to assess the potential for using these methods at the control stages during production of heterologous antirabies immunoglobulin obtained from equine serum. The search for cutting-edge highly sensitive in vitro control methods that could compete with the biological method, which is the main method used in antirabies immunoglobulin control, is an important prerequisite for improvement of the production technology and the quality of antirabies medicines. The study demonstrated that the following test methods can be used in the production of antirabies immunoglobulin: fluorescent antibody technique, enzyme-linked immunosorbent assay, cell culture methods, atomic force microscopy, and flow cytometry. These methods could be used alone or as an alternative to the biological method in white mice. These methods were chosen because of their high sensitivity, specificity, rapid and easy implementation, cost-effectiveness, and automatic recording of test results.

Humans need a dietary source of glucose, which is the preferred substrate for generating metabolic energy in the brain, red blood cells, kidney and reproductive tissues. Starch is the main source of dietary glucose for most humans. The ability to assess the glycemic (i.e., blood glucose-raising) potential of starchy foods is highly relevant to nutrition and health, as the inability to maintain the blood glucose concentration within normal limits is a risk for diet-related illnesses and obesity. In vivo tests assess the glycemic impact of starchy foods in terms of a glycemic index (GI). However, GI testing has inherent limitations and in vitro model systems are used widely as a proxy for assessing glycemic impact; these in vitro methods are important for assessing starch digestibility and the quality of carbohydrates in many applications. This review will focus on in vitro methods for measuring starch digestibility. Starch as the main glycemic carbohydrate in the human diet, how starch is broken down enzymically, and how GI values are determined are first considered briefly, before discussing methods and advantages of in vitro testing. The limitations, which as the literature shows are not always appreciated and can lead to over-interpretation of research data, are also considered. The importance of in vitro methodology for assessing the carbohydrate quality of foods is clear, but there is a need to standardise procedures to facilitate comparability of studies conducted at different times and in different laboratories. [ABSTRACT FROM AUTHOR]

Bioavailability assessment in the development phase of a drug product is vital to reveal the disadvantageous properties of the substance and the possible technological interventions. However, in vivo pharmacokinetic studies provide strong evidence for drug approval applications. Human and animal studies must be designed on the basis of preliminary biorelevant experiments in vitro and ex vivo. In this article, the authors have reviewed the recent methods and techniques from the last decade that are in use for assessing the bioavailability of drug molecules and the effects of technological modifications and drug delivery systems. Four main administration routes were selected: oral, transdermal, ocular, and nasal or inhalation. Three levels of methodologies were screened for each category: in vitro techniques with artificial membranes; cell culture, including monocultures and co-cultures; and finally, experiments where tissue or organ samples were used. Reproducibility, predictability, and level of acceptance by the regulatory organizations are summarized for the readers.

The alarming increase in the number of advanced-stage prostate cancer cases with poor prognosis has led to a search for innovative methods of treatment. In response to the need for implementation of new and innovative methods of cancer tissue therapy, we studied photodynamic action in excised prostate tissue in vitro as a model for photodynamic therapy. To ascertain the effects of photodynamic action in prostate tissue, Rose Bengal (0.01 to 0.05 mM) was used as a photosensitizer in the presence of oxygen and light to generate singlet oxygen in tissues in vitro. Five preset concentrations of Rose Bengal were chosen and injected into prostate tissue samples (60 samples with 12 replications for each RB concentration) that were subsequently exposed to 532 nm light. The effects of irradiation of the Rose Bengal infused tissue samples were determined by histopathological analysis. Histopathological examination of prostate samples subjected to photodynamic action revealed numerous changes in the morphology of the neoplastic cells and the surrounding tissues. We conclude that the morphological changes observed in the prostate cancer tissues were a result of the photogeneration of cytotoxic singlet oxygen. The tissue damage observed post photodynamic action offers an incentive for continued in vitro investigations and future in vivo clinical trials. [ABSTRACT FROM AUTHOR]

Breeding for new ornamental varieties is quite often a profitable occupation in the world. Worldwide breeding activities have generated a massive amount of literature. All present day ornamental varieties have evolved through bud sports, open pollinated interspecific crosses, open pollinated intraspecific crosses, planned crosses, natural chromosomal changes, chromosomal manipulations, induced mutations and using new breeding techniques like management of chimera, in vitro mutation, molecular approaches, somaclonal variations etc. The breeders have modified and improved these techniques on need basis to achieve targeted and directional changes. All work on ornamental breeding and development of new varieties are the results of concerted efforts of scientific community, nurserymen, breeders, amateur growers and flower lovers. It is important to highlight the factual account of the early work, genuine drawback of fulfillments and success of ornamental breeding. It is now needful to assess the present status of each technology developed after extensive research activities conducted by different research institutions. Breeders will be familier with the capability and constraints of different techniques and select the proper experimental approach which is most suitable and profitable to achieve their goals. This review provides an indeepth knowledge base for accurate designing of future breeding strategies in the field of ornamentals. The entire breeding system of ornamentals have been very simply reviewed and cited with very common examples so that even a new breeder can follow and select the most suitable techniques for developing the new varieties with desired traits of commercial ornamental species. [ABSTRACT FROM AUTHOR]

The site of action of centrally acting drugs lies inside the brain and therefore, needs to reach the brain to exert their therapeutic efficacy. Discovery and development process of such types of drugs demands their quantification in brain to establish the dose, study pharmacokinetics, pharmacodynamics, and optimize the overall efficacy. Moreover, some drugs of other categories also have potential to cross blood–brain barrier resulting in various adverse events by acting centrally. However, the collection of a matrix to analyze the amount of drugs present in brain is highly challenging. In this review, we have summarized different bioanalytical strategies to quantitate drugs inside the brain. A detailed discussion on various in vivo and in vitro techniques for monitoring drugs inside the brain has been incorporated. In addition, various sampling techniques have been discussed in brief with case studies. Therefore, this review can guide the researcher to choose appropriate bioanalytical techniques for analyzing drugs in brain depending upon the specific need and quantification threshold considering the commonly associated difficulties of the methods. [ABSTRACT FROM AUTHOR]

Detailed assessment of the biotransformation of compounds and the activity of their metabolites is an extremely important element in the safety evaluation of a substance, both in preclinical and clinical studies. It should be noted that the metabolite may differ from the parent compound in terms of physicochemical properties and consequently pharmacological and toxicological properties. the purpose of the biotransformation of xenobiotics is to increase their hydrophilicity, which allows excretion in the urine. the metabolites of biotransformation phase I show undesirable pharmacological or toxic effects much more often. according to U.S. Food and Drug administration (FDa) guidance, if a metabolite in the human body accounts for more than 10% of the total amount of metabolites, its safety should be thoroughly assessed. Metabolites can interact more strongly or weakly, or to the same degree, with the same or a different molecular target as the parent compound. they can also display non-specific effects by, for example, damaging macromolecules (e.g. proteins, nucleic acids) in the way that free radicals do. In the case of new psychoactive substances (NPSs), the pharmacological properties, including metabolism, are largely unknown until they are traded illegally and their users begin to appear in departments of toxicology or forensic medicine. It is in this way that the activity profile and probable metabolic pathways of NPSs are determined. to determine the metabolites of NPSs is also an important toxicological skill in the forensic testing of biological samples (blood/urine/tissue) collected from victims, in which metabolites, not parent compounds, are usually found. Using examples from the authors' research and the available literature, the article aims to present alternative methods of metabolism testing for NPSs. In vitro methods (application of microsomes, S9 fraction, hepatocytes, cytosol) are discussed and comparisons are made between the results of in vivo tests on animals and analyses of autopsy material. the experiments and the literature review demonstrate that by using in vitro methods the metabolism of NPSs can be predicted with high probability. By improving existing methods of metabolism research and creating new and alternative ones it will be possible to better understand metabolic pathways and better identify the NPS metabolites formed in the human body. this will contribute not only to the development of better methods of treating NPS poisoning, but will also be of use when compiling forensic and medical reports for the judiciary. [ABSTRACT FROM AUTHOR]

Inter-species differences in toxicodynamics are often a critical source of uncertainty in safety evaluations and typically dealt with using default adjustment factors. In vitro studies that use cells from different species demonstrated some success for estimating the relationships between life span and/or body weight and sensitivity to cytotoxicity; however, no apparent investigation evaluated the utility of these models for risk assessment. It was hypothesized that an in vitro model using dermal fibroblasts derived from diverse species and individuals might be utilized to inform the extent of inter-species and inter-individual variability in toxicodynamics. To test this hypothesis and characterize both inter-species and inter-individual variability in cytotoxicity, concentration-response cytotoxicity screening of 40 chemicals in primary dermal fibroblasts from 68 individuals of 54 diverse species was conducted. Chemicals examined included drugs, environmental pollutants, and food/flavor/fragrance agents; most of these were previously assessed either in vivo or in vitro for inter-species or inter-individual variation. Species included humans, the typical preclinical species and representatives from other orders of mammals and birds. Data demonstrated that both inter-species and inter-individual components of variability contribute to the observed differences in sensitivity to cell death. Further, it was found that the magnitude of the observed inter-species and inter-individual differences was chemical-dependent. This study contributes to the paradigm shift in risk assessment from reliance on in vivo toxicity testing to higher-throughput in vitro or alternative approaches, extending the strategy to replace use of default adjustment factors with experimental characterization of toxicodynamic inter-individual variability and to also address toxicodynamic inter-species variability. [ABSTRACT FROM AUTHOR]

The main objective of the present research was to review the studies that look into ways of improving production of anti-rabies immunoglobulin in terms of bioethical principles: ensuring patients’ right to receive quality pharmaceutical care, and compliance with the 3R principles. At the same time, compliance with bioethical principles should contribute to the improvement of production technology and the product quality, which is especially important for antirabies immunoglobulin due to the existing high demand for it. The paper analyses the current trends in avoidance of animal use in the production of rabies immunoglobulin. It summarises the main methods of production of serum products for post-exposure prophylaxis of rabies. The example of heterologous rabies immunoglobulin is used to substantiate the need to improve quality control of drugs by following the 3R principles. The paper highlights the potential use of cell cultures for determination of rabies immunoglobulin specific activity. The authors formulated the objectives that include development and use of pyrogen detection methods which do not involve animal use and are consistent with the current pharmacopoeial standards. They assessed the possibility and feasibility of removing Abnormal Toxicity Test for heterologous rabies immunoglobulin in accordance with the current international trends. The formulated objectives imply optimisation of production of heterologous rabies immunoglobulin in Russia in order to improve the quality of antirabies measures. The objectives include higher production volumes to ensure the availability of the product to patients, reduction of the product reactogenicity by using cell-culture technologies for obtaining rabies antigen, as well as development and implementation of in vitro test methods for product quality control in terms of Specific activity, Pyrogenicity, and Abnormal toxicity

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

Summary: The diagnosis of biofilms continues to be a challenge, and there is no standardized protocol for such a diagnosis in clinical practice. In addition, some proposed methodologies are expensive to require significant amounts of time and a high number of trained staff, making them impracticable for clinical practice. In recent years, mass spectrophotometry/matrix‐assisted laser desorption ionization time of flight (MALDI‐TOF) has been applied it in biofilm studies. However, due to several problems and limitations of the technique, MALDI‐TOF is far from being the gold standard for identifying biofilm formation. The omics analysis may prove to be a promising strategy for the diagnosis of biofilms in clinical laboratories since it allows the identification of pathogens in less time than needed for conventional techniques and in a more specific manner. However, omic tools are expensive and require qualified technical expertise, and an analysis of the data obtained needs to be careful not to neglect subpopulations in the biofilm. More studies must therefore be developed for creating a protocol that guarantees rapid biofilm identification, ensuring greater chances of success in infection control. This review discusses the current methods of microbial biofilm detection and future perspectives for its diagnosis in clinical practice. [ABSTRACT FROM AUTHOR]

Proteins are large molecules that play essential roles in all living organisms. In most molecular processes in each cell, proteins usually do not function alone but through physiological interactions with various ligands. The most common interacting molecules for proteins are other proteins, and they act together by protein-protein interactions (PPIs) to create larger protein complexes. The impairment of physiological PPIs or establishing PPIs with pathological proteins often leads to the development of diseases. To bring insights on the knowledge about the physiological functions of proteins in biological processes, and to understand the development and pathogenesis of diseases, numerous qualitative and quantitative methods have been developed. In this review, we summarize the most commonly used methods for studying PPIs, and discuss their advantages and drawbacks. [ABSTRACT FROM AUTHOR]

Phenolic compounds have demonstrated several in vitro beneficial properties by acting as antioxidant and pro-oxidant agents. This chapter approaches the relationship among oxidative stress, cancer, phenolic compounds and antiproliferative activity. Moreover, it discusses in vitro techniques and their biological applications, regarding cell viability and intracellular measure of reactive oxygen assays. The in vitro methods are important tools for screening and understanding the pathways involved on antiproliferative and antioxidant/pro-oxidant effects of phenolic compounds. These findings open avenues for the development of innovative food, chemical structures, technological applications and future perspectives in this research field. [ABSTRACT FROM AUTHOR]

Newly synthesized chemicals are being introduced into the environment without undergoing proper toxicological evaluation, particularly in terms of their effects on the vulnerable neurodevelopment. Thus, it is important to carefully assess the developmental neurotoxicity of these novel environmental contaminants using methods that are closely relevant to human physiology. This study comparatively evaluated the potential developmental neurotoxicity of 19 prevalent environmental chemicals including neonicotinoids (NEOs), organophosphate esters (OPEs), and synthetic phenolic antioxidants (SPAs) at environment-relevant doses (100 nM and 1 μM), using three commonly employed in vitro neurotoxicity models: human neural stem cells (NSCs), as well as the SK-N-SH and PC12 cell lines. Our results showed that NSCs were more sensitive than SK-N-SH and PC12 cell lines. Among all the chemicals tested, the two NEOs imidaclothiz (IMZ) and cycloxaprid (CYC), as well as the OPE tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), generated the most noticeable perturbation by impairing NSC maintenance and neuronal differentiation, as well as promoting the epithelial-mesenchymal transition process, likely via activating NF-κB signaling. Our data indicate that novel NEOs and OPEs, particularly IMZ, CYC, and TDCIPP, may not be safe alternatives as they can affect NSC maintenance and differentiation, potentially leading to neural tube defects and neuronal differentiation dysplasia in fetuses. [Display omitted] • The developmental neurotoxicity of 19 emerging environmental chemicals was assessed. • Neural stem cells were more sensitive to the chemicals than the SK-N-SH and PC12 cell lines. • Among the chemicals tested, IMZ, CYC, and TDCIPP exhibited the most adverse effects. • IMZ, CYC, and TDCIPP impaired NSC maintenance and differentiation, likely via NF-κB. • This study provides valuable insights for the safe use of emerging environmental chemicals. [ABSTRACT FROM AUTHOR]

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y.

The alarming increase in the number of advanced-stage prostate cancer cases with poor prognosis has led to a search for innovative methods of treatment. In response to the need for implementation of new and innovative methods of cancer tissue therapy, we studied photodynamic action in excised prostate tissue in vitro as a model for photodynamic therapy. To ascertain the effects of photodynamic action in prostate tissue, Rose Bengal (0.01 to 0.05 mM) was used as a photosensitizer in the presence of oxygen and light to generate singlet oxygen in tissues in vitro. Five preset concentrations of Rose Bengal were chosen and injected into prostate tissue samples (60 samples with 12 replications for each RB concentration) that were subsequently exposed to 532 nm light. The effects of irradiation of the Rose Bengal infused tissue samples were determined by histopathological analysis. Histopathological examination of prostate samples subjected to photodynamic action revealed numerous changes in the morphology of the neoplastic cells and the surrounding tissues. We conclude that the morphological changes observed in the prostate cancer tissues were a result of the photogeneration of cytotoxic singlet oxygen. The tissue damage observed post photodynamic action offers an incentive for continued in vitro investigations and future in vivo clinical trials.

Phytoplankton photosynthesize in surface waters, exporting organic carbon to depth through the biological pump. Quantifying productivity and the export of carbon is important to understanding the global carbon cycle and predicting its future changes. An issue in quantifying rates is that the many existing methods are not all equivalent, making comparisons between studies using different methods challenging. Our goal is to compare in situ and in vitro methods in order to identify where methods agree in the NE subarctic Pacific. During this study, we measured productivity using two in situ methods (oxygen/argon ratio and triple oxygen isotope mass balance approaches) and four in vitro methods (13C, NO3−15, NH4+15, and H218O uptake rates through incubations), and compared the results with one satellite‐based productivity estimate. The in situ carbon export method was consistently higher than the in vitro method, likely due to dissolved organic matter release not included in our incubation measurements. Upwelling bringing low‐O2 water to the surface and the interaction between bloom dynamics and a method's inherent time of integration cause outliers from the relationship. In contrast, in situ and in vitro methods for estimating gross primary production were consistent across a wide range in rates. We find that chlorophyll‐a concentration is strongly related to many of our measured rates. Satellite estimates of primary production are consistently different from 13C incubations. Our identification of consistent trends and causes for disagreement will allow observations from one method to be converted to another, facilitating future comparisons across studies. Plain Language Summary: Phytoplankton in the surface ocean photosynthesize, taking up carbon dioxide and producing oxygen. Phytoplankton form the base of the food web and play an important role in transferring carbon to the deep ocean, away from the atmosphere. Quantifying how fast phytoplankton grow and how much carbon makes it out of the surface waters will help predict future climate. One issue preventing accurate quantification is that different methods disagree, making it difficult to compare between studies. We find there are consistent differences between marine productivity methods in the NE Pacific. Incubations that remove water from the environment tend to have lower estimates for carbon transferred out of surface waters than methods that measure the natural abundance of gases. At higher rates, methods are less consistent. In contrast, both method types estimating gross photosynthesis rates are consistent over a wide range of rates. We are able to establish consistent relationships between an incubation method and other methods, including satellite estimates and chlorophyll concentrations. The consistent agreement between rate measurements expands comparisons that can be made between studies that use different methods, allowing for a better understanding of phytoplankton's role in controlling carbon processes. Key Points: We identify consistent relationships among six productivity rate methods as well as with chlorophyll‐a in the NE subarctic Pacific regionDissolved organic matter release likely causes incubation‐based carbon export to be consistently low compared to the in situ estimateUpwelling events and the timing of bloom dynamics influence outliers from the consistent relationships [ABSTRACT FROM AUTHOR]

Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells/tissues of human origin. [ABSTRACT FROM AUTHOR]

Abstract Background Improving our understanding of cancer and other complex diseases requires integrating diverse data sets and algorithms. Intertwining in vivo and in vitro data and in silico models are paramount to overcome intrinsic difficulties given by data complexity. Importantly, this approach also helps to uncover underlying molecular mechanisms. Over the years, research has introduced multiple biochemical and computational methods to study the disease, many of which require animal experiments. However, modeling systems and the comparison of cellular processes in both eukaryotes and prokaryotes help to understand specific aspects of uncontrolled cell growth, eventually leading to improved planning of future experiments. According to the principles for humane techniques milestones in alternative animal testing involve in vitro methods such as cell-based models and microfluidic chips, as well as clinical tests of microdosing and imaging. Up-to-date, the range of alternative methods has expanded towards computational approaches, based on the use of information from past in vitro and in vivo experiments. In fact, in silico techniques are often underrated but can be vital to understanding fundamental processes in cancer. They can rival accuracy of biological assays, and they can provide essential focus and direction to reduce experimental cost. Main body We give an overview on in vivo, in vitro and in silico methods used in cancer research. Common models as cell-lines, xenografts, or genetically modified rodents reflect relevant pathological processes to a different degree, but can not replicate the full spectrum of human disease. There is an increasing importance of computational biology, advancing from the task of assisting biological analysis with network biology approaches as the basis for understanding a cell’s functional organization up to model building for predictive systems. Conclusion Underlining and extending the in silico approach with respect to the 3Rs for replacement, reduction and refinement will lead cancer research towards efficient and effective precision medicine. Therefore, we suggest refined translational models and testing methods based on integrative analyses and the incorporation of computational biology within cancer research.

Food companies should comply with the requirements of a zero‐waste concept to adapt to the circular economy requirements. In fruit companies, usually seeds are discarded without proper utilization and extraction of the bioactive compounds. Fruit seeds are sources of chemical compounds that can be extracted, studied, and applied in high value‐added products. Thus, in this work the experimental conditions for the water extraction of phenolic compounds from camu‐camu (Myrciaria dubia) seed coat were optimized using a central composite design and the desirability function. Total phenolic content (TPC), and condensed tannins (CT), DPPH radical scavenging activity, ferric reducing antioxidant capacity (FRAP), Folin‐Ciocalteu reducing capacity (FCRC), and Cu2+chelating ability were assessed. Seed coat extracted for 51.1 min using a 1:34.1 solid:liquid ratio was the optimal condition to extract 6,242 mg gallic acid equivalent (GAE)/100 g of TPC and 695 mg catechin equivalent (CE)/100 g of CT. The optimized extract displayed free‐radical scavenging activity, reducing properties and ability to chelate Cu2+, and inhibited the growth of Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Salmonella Enteritidis, Bacillus cereus, and Staphylococcus aureus. Additionally, the lyophilized water extract inhibited the in vitro activity of α‐amylase, α‐glucosidase, and angiotensin converting enzyme and showed cytotoxic effects towards Caco‐2, A549, and HepG2 cancer cells, but no cytotoxicity towards IMR90 cells. Vescalagin, castalagin, and 3,4‐dihydroxybenzoic acid were the major phenolic compounds identified in the optimized extract. In conclusion, the optimized camu‐camu seed coat water extract is a rich source of phenolic compounds with antioxidant, antidiabetic, antihypertensive, and antiproliferative effects. Practical Application: Camu‐camu fruit pulp and seeds have been studied for their phenolic composition and bioactivity. However, seeds are usually discarded and represent an environmental problem in South American countries. We presented a methodological overview on the extraction optimization of the phenolic compounds from camu‐camu seed coat and studied the bioactivity of the optimized extract using chemical, enzymatic, and cell‐based experiments. Results can be used by camu‐camu processors to obtain a phenolic‐rich extract for industrial applications, without any further processing. [ABSTRACT FROM AUTHOR]

The variable taxa components of human gut microbiota seem to have an enormous biotechnological potential that is not yet well explored. To investigate the usefulness and applications of its biocompounds and/or bioactive substances would have a dual impact, allowing us to better understand the ecology of these microbiota consortia and to obtain resources for extended uses. Our research team has obtained a catalogue of isolated and typified strains from microbiota showing resistance to dietary contaminants and obesogens. Special attention was paid to cultivable Bacillus species as potential next-generation probiotics (NGP) together with their antimicrobial production and ecological impacts. The objective of the present work focused on bioinformatic genome data mining and phenotypic analyses for antimicrobial production. In silico methods were applied over the phylogenetically closest type strain genomes of the microbiota Bacillus spp. isolates and standardized antimicrobial production procedures were used. The main results showed partial and complete gene identification and presence of polyketide (PK) clusters on the whole genome sequences (WGS) analysed. Moreover, specific antimicrobial effects against B. cereus, B. circulans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Serratia marcescens, Klebsiella spp., Pseudomonas spp., and Salmonella spp. confirmed their capacity of antimicrobial production. In conclusion, Bacillus strains isolated from human gut microbiota and taxonomic group, resistant to Bisphenols as xenobiotics type endocrine disruptors, showed parallel PKS biosynthesis and a phenotypic antimicrobial effect. This could modulate the composition of human gut microbiota and therefore its functionalities, becoming a predominant group when high contaminant exposure conditions are present.

Plant extracts and essential oils (EOs) are characterized by their antibacterial properties against various bacterial pathogens, including staphylococci. Some strains of these bacteria are resistant against the adverse effects of the environment including antibiotics, e. g. methicillin-resistant Staphylococcus aureus (MRSA). EOs alone cannot substitute for antibiotics but their treatment may be useful to intensify and strengthen the effects of antibiotics on pathogenic staphylococci. In this work, we tested the antibacterial effects of the essential oils of Mentha species with menthol as one of the effective substances against different strains of S. aureus. Two in vitro methods were used, the qualitative disc diffusion assay and the quantitative minimal inhibitory concentration (MIC) of selected essential oils. Peppermint oil from Mentha piperita, spearmint oil from Mentha spicata var. crispa and cornmint oil from Mentha arvensis were tested in this study against the various strains of Staphylococcus aureus, including methicillin resistant Staphylococcus aureus (MRSA). The oils were dissolved in DMSO (dimethyl sulfoxide) and diluted at the following ratios: 1 : 1, 1 : 2, 1 : 5, and 1 : 10. Based on the results determined by the agar disc diffusion test, the highest antibacterial properties were observed in spearmint oil against S. aureus CCM 4223 at 1 : 2 ratio where the inhibition zone varied at a range of 35.67 ± 6.81 mm. We determined also the MIC of all the oils where concentrations of the oils were as follows: 1 %; 0.5 %; 0.25 %; 0.125 % and 0.0625 %. The lowest concentrations of essential oils that possessed inhibitory effects on the growth of S. aureus varied between 0.125 % and 0.25 %.