Ochratoxin A induces hepatic and renal toxicity in mice through increased oxidative stress, mitochondrial damage, and multiple cell death mechanisms.

Ochratoxin A (OTA) is a widespread food toxin produced by Aspergillus ochraceus and other molds. In this study, we developed and established acute OTA toxicity conditions in mice, which received daily oral doses of OTA between 0.5 up to 8 mg/kg body weight up to 7 days and were subjected to histological and biochemical analysis to characterize renal and hepatic damage. Oral administration of OTA for 7 days resulted in loss of body weight in a dose-dependent manner and increased the levels of serum biomarkers of hepatic and renal damage. The kidney was more sensitive to OTA-induced damage than the liver. In addition to necrosis, OTA induced hepatic and renal apoptosis in dose- and time-dependent manners. Especially, a high dose of OTA (8 mg/kg body weight) administered for 7 days led to necroptosis in both liver and kidney tissues. OTA dose-dependently increased the oxidative stress levels, including lipid peroxidation, in the liver and kidneys. OTA disrupted mitochondrial dynamics and structure in hepatic and renal cells, leading to the dysregulation of mitochondrial homeostasis. OTA increased transferrin receptor 1 and decreased glutathione peroxidase 4 levels in a dose- and time-dependent manner. These results suggest the induction of ferroptosis. Collectively, this study highlighted the characteristics of acute OTA-induced hepatic and renal toxicity in mice in terms of oxidative stress, mitochondrial damage, and multiple cell death mechanisms, including necroptosis and ferroptosis.

Utility of a Multilevel Modeling Approach to Investigate Differences in Isolation Frequency of Fusarium culmorum in Agricultural Soil Across the Inland Pacific Northwest.

The plant pathogen Fusarium culmorum represents an inoculum source capable of contaminating grains with deoxynivalenol in the Inland Northwest region of the United States. A multilevel modeling approach utilizing varying intercepts for different sampling quadrats, fields, and iterations in the dataset was performed to characterize differences in isolation frequency of F. culmorum collected during a 2-year soil survey. Differences in the isolation frequency of F. culmorum varied the most by sampled field followed by quadrat and iteration, respectively. Higher relative elevation within the sampling region of a field limited the amount of F. culmorum recovered. The effect of annual climate variables was investigated using combinations of single-variable and multivariable model equations with linear and polynomial terms. The same data analysis approach was applied to an external dataset of F. culmorum isolation frequencies in grains from fields across eastern Australia. These results represent a case study for investigating variability within datasets containing overdispersed fungal counts and incorporating climate summaries as predictor variables.

Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Development of a stir bar sorptive extraction method for analysis of ochratoxin A in beer.

A new, simple, and sensitive method was developed for extraction of ochratoxin A (OTA) in beer combined with HPLC-fluorescence detector. Samples were extracted by stir bar sorptive extraction (SBSE) followed by liquid desorption using commercially available Twister EG-Silicone. The main parameters influencing SBSE, including phase ratio, extraction time, stirring speed, ionic strength, organic modifier, pH, temperature, desorption mode, desorption solvent, and desorption time were optimized. Under the optimal conditions, assay was performed on 4 mL of samples acidified at pH 1.5. The samples were extracted for 180 min at a stirring speed of 800 rpm followed by desorption of analyte using 1 mL methanol under sonication for 45 min. The extract was evaporated at 50 degrees C under a gentle nitrogen stream, then redissolved in 200 microL of methanol-water (50 + 50, v/v). After each use, the stir bar was cleaned by sonication in methanol for 30 min three times. The method provided good linearity of the calibration curve with coefficients of determination greater than 0.999. Recoveries of OTA were greater than 83% with RSD lower than 10%, and LOD of OTA in beer was 0.64 ng/L. This method was applied to determine OTA in 19 beer samples. OTA was detected in 12 samples (63%) in the range of 0.01-0.27 ng/mL.

Practical Strategies to Reduce Ochratoxin A in Foods.

Ochratoxin A (OTA), a potent nephrotoxin, is one of the most deleterious mycotoxins, with its prevalence in agricultural crops and their processed foods around the world. OTA is a major concern to food safety, as OTA exposure through dietary intake may lead to a significant level of accumulation in the body as a result of its long half-life (about 35 days). Its potent renal toxicity and high risk of exposure as well as the difficulty in controlling environmental factors OTA production has prompted the need for timely information on practical strategies for the food industry to effectively manage OTA contamination during food processing. The effects of various food processes, including both nonthermal and thermal methods, on the reduction in OTA were summarized in this review, with emphasis on the toxicity of residual OTA as well as its known and unknown degradation products. Since complete removal of OTA from foodstuffs is not feasible, additional strategies that may facilitate the reduction in OTA in food, such as adding baking soda and sugars, was also discussed, so that the industry may understand and apply practical measures to ensure the safety of its products destined for human consumption.

Food safety training needs at evacuation shelters operated by faith-based organizations.

The authors conducted a survey to identify food safety training needs at evacuation shelters operated by faith-based organizations (FBOs) in four hurricane-prone states. Five thousand randomly selected FBO leaders were asked questions about their food safety attitudes and food handling practices at evacuation shelters. Descriptive statistics and multivariate analysis of variance were calculated to summarize and prioritize the responses. Results from 138 leaders revealed that on average, 590 +/- 4,787 evacuees were served for 36 +/- 72 days at FBO-operated shelters. Only 19.6% felt they were well prepared for the shelter. Only 5.8% had professional food preparation staff and many accepted hot (47.8%) and cold (37%) prepared food donations. Some lacked adequate refrigerator (18.8%) or freezer (16.7%) spaces, but 40% kept hot food leftovers for later use. The majority did not provide food safety training before opening the shelters (73.2%), yet 76.9% said they will provide food to evacuation shelters again. The results show a need for food safety training and specific strategies for training at FBOs.

Protective Effect of alpha-Tocopherol Against Ochratoxin A in Kidney Cell Line HK-2.

Food safety is a top priority for the protection of infants and young children. Ochratoxin A (OTA) is an emerging concern due to its high toxicity and occurrence in a wide range of agricultural crops and their derived food products including those foods and snacks destined for infants and young children. OTA is considered as a possible human carcinogen, and its main target organ is the kidney. The objective of this study was to investigate the protective effect of α-tocopherol against oxidative stress induced by OTA using human proximal tubule epithelial cells (HK-2). OTA showed dose-dependent increase in cytotoxicity (IC50 = 161 nM, p < 0.05) at 48 h, while treatment up to 2 mM α-tocopherol did not change cell viability. Levels of the reduced form of glutathione (GSH) were decreased with α-tocopherol treatment, although the ratio of the oxidative form (GSSG) to GSH remained the same. Among several genes associated with oxidative stress, expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione reductase (GSR), and kidney injury molecule-1 (KIM-1) were significantly up-regulated by OTA treatment. CAT and GSR showed decreased expression at 0.5-2 mM α-tocopherol and OTA at IC50 value, KIM-1 was decreased at 0.5 mM α-tocopherol and OTA at IC50 value, and nuclear factor erythroid 2-related factor 2 (Nrf2) was decreased at 0.5-1 mM α-tocopherol and OTA at IC50 value. In addition, the levels of malondialdehyde (MDA) were increased significantly by OTA while significantly decreased by α-tocopherol. The results show that α-tocopherol may alleviate potential OTA-induced renal damage and oxidative stress through reducing cytotoxicity and enhancing the antioxidant defense systems.

High-performance liquid chromatography using diode array detector and fluorescence detector for hydrogen peroxide analysis in processed fishery foods.

Hydrogen peroxide (H2O2) is a food additive for bleaching and sterilization, owing to its strong oxidizing effect. The current study aimed to develop analytical methods to detect trace amounts of residual H2O2 in diverse foods using high-performance liquid chromatography (HPLC) equipped with diode array detector (DAD) or fluorescence detector (FLD). The vanadium(V)-peroxo complex, derived from the reaction of H2O2 with ammonium metavanadate, was used for the detection of H2O2 with DAD. H2O2 was indirectly analyzed using FLD via the detection of 7-hydroxycoumarin, derived by Fenton reaction, followed by verification using liquid chromatography with tandem mass spectrometry analysis. Both the detection methods showed good linearity with R2 > 0.997. Limit of detection and limit of quantification were 0.30 and 0.91 mg/L (8.82 and 26.76 µM) with HPLC-DAD and 0.001 and 0.003 mg/L (0.03 and 0.09 µM) with HPLC-FLD, respectively. Applicability of both the methods was successively tested through sample analysis.

Ochratoxin A and its reaction products affected by sugars during heat processing.

Ochratoxin A (OTA) is a nephrotoxin produced by many species in two fungal genera of Aspergillus and Penicillium under virtually all agricultural environments. Hence, OTA occurs frequently in agricultural commodities and their downstream products worldwide. In this study, thermal stability of OTA in the presence of sugars commonly added to food products including glucose, fructose, and sucrose was investigated by analyzing their reaction products with HPLC-FLD and LC-MS/MS. Samples were heated at three different temperatures (100, 125, and 150 °C) in 10-min intervals for up to 60 min in the absence of food matrix. Analysis showed increased OTα and OTα-amide and decreased OTA isomer (14-R-OTA) formation when OTA was heated with sugars. Among the sugars tested, adding fructose resulted in significantly lower OTA levels than glucose, sucrose, or no sugar added control. Addition of fructose also shifted OTA degradation product profile to less toxic OTα-amide, instead of OTA isomer which has similar toxicity to OTA. These results suggest that added sugars influenced the levels of OTA and its degradation products formed during thermal processing, and may provide a means to reduce the toxicity of OTA in food.

Reduction of Ochratoxin A during the Preparation of Porridge with Sodium Bicarbonate and Fructose.

Ochratoxin A (OTA) is a potential human carcinogen that poses a significant concern in food safety and public health. OTA has been found in a wide variety of agricultural commodities, including cereal grains. This study investigated the reduction of OTA during the preparation of rice- and oat-based porridge by a simulated indirect steam process. The effects of sodium bicarbonate (NaHCO3) and fructose on the reduction of OTA were also investigated. During the processing, OTA in rice- and oat-porridge was decreased by 59% and 14%, respectively, from initial OTA artificially added at 20 µg/kg (dry weight basis). When 0.5% and 1% of sodium bicarbonate were added to rice porridge, increased reduction of OTA was observed as 78% and 68%, respectively. The same amounts of added sodium bicarbonate also further reduced OTA in oat porridge to 58% and 72%, respectively. In addition, increased reduction of OTA in the presence of fructose was observed. A combination of the two, i.e., 0.5% sodium bicarbonate and 0.5% fructose, resulted in a 79% and 67% reduction in rice porridge and oat porridge, respectively. These results indicate that indirect steaming may effectively reduce OTA in preparation of porridge-type products, particularly when sodium bicarbonate and/or fructose are added.

Ochratoxin A Induces Oxidative Stress in HepG2 Cells by Impairing the Gene Expression of Antioxidant Enzymes.

Ochratoxin A (OTA) is a mycotoxin frequently found in raw and processed foods. While it is considered a possible human carcinogen, the mechanism of action remains unclear. OTA has been shown to be hepatotoxic in both in vitro and in vivo models and oxidative stress may be one of the factors contributing to its toxicity. Hence, the effect of OTA on human hepatocellular carcinoma, HepG2 cells, was investigated on oxidative stress parameters. The cytotoxicity of OTA on HepG2 was time- and dose-dependent within a range between 0.1 and 10 µM; while 100 µM of OTA increased the intracellular reactive oxygen species (ROS) in a time-dependent manner. Additionally, the levels of glutathione (GSH) were increased by 9.7% and 11.3% at 10 and 100 nM of OTA, respectively; while OTA at 100 µM depleted GSH by 40.5% after 24 h exposure compared with the control. Finally, the mRNA level of catalase (CAT) was downregulated by 2.33-, 1.92-, and 1.82-fold after cells were treated with 1, 10, and 10 µM OTA for 24 h, respectively; which was linked to a decrease in CAT enzymatic activity. These results suggest that oxidative stress is involved in OTA-mediated toxicity in HepG2 cells.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress.

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

New Analytical Approach for The Determination of Calcium Phosphate Dibasic and Tribasic in Processed Food by Comparison of Ion Chromatography With High-Performance Liquid Chromatography.

An analytical method to measure solubilized orthophosphate ions (HPO42- and PO43- ) from the water-insoluble food additives calcium phosphate dibasic (DCP) and calcium phosphate tribasic (TCP) in processed foods was optimized by comparing ion chromatography (IC) coupled with DS6 conductivity detector (Cond.) and high-performance liquid chromatography (HPLC) with Evaporative light scattering detector (ELSD) methods. The ion-pairing HPLC method could analyze calcium and phosphate ions successively. However, this method exhibited low reproducibility after approximately 48 hours of measurements. The IC method was established as an effective method of measuring orthophosphate ions with high reproducibility using distilled water and KOH solution as the mobile phase with a Dionex column. Matrix-based limit of detections (LOD) and limit of quantifications (LOQ) for snacks and cereals were estimated in the range of 0.01-0.91 µg/mL and 0.21-2.74 µg/mL, respectively. In inter-day and intra-day tests, the calculated precision (%RSD) and accuracy (recovery %) ranged from 0.5% to 6.6% and 82% to 117%, respectively, in both food samples. The levels of DCP or TCP could be analyzed in various positive food samples, and the developed IC method demonstrated good applicability in the analysis of DCP and TCP in collected processed foods.

Ochratoxin A-Induced Hepatotoxicity through Phase I and Phase II Reactions Regulated by AhR in Liver Cells.

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of the genera Aspergillus and Penicillium. OTA exists in a variety of foods, including rice, oats, and coffee and is hepatotoxic, with a similar mode of action as aflatoxin B1. The precise mechanism of cytotoxicity is not yet known, but oxidative damage is suspected to contribute to its cytotoxic effects. In this study, human hepatocyte HepG2 cells were treated with various concentrations of OTA (5-500 nM) for 48 h. OTA triggered oxidative stress as demonstrated by glutathione depletion and increased reactive oxygen species, malondialdehyde level, and nitric oxide production. Apoptosis was observed with 500 nM OTA treatment. OTA increased both the mRNA and protein expression of phase I and II enzymes. The same results were observed in an in vivo study using ICR mice. Furthermore, the relationship between phase I and II enzymes was demonstrated by the knockdown of the aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) with siRNA. Taken together, our results show that OTA induces oxidative stress through the phase I reaction regulated by AhR and induces apoptosis, and that the phase II reaction is activated by Nrf2 in the presence of oxidative stress.

Probabilistic risk model of norovirus transmission during handling and preparation of fresh produce in school foodservice operations.

Human noroviruses (NoV) are recognized worldwide as important pathogens and the primary cause of foodborne disease outbreaks from contaminated food in the U.S. They are often transmitted by infected food handlers manipulating foods during preparation, such as fresh fruits and vegetables. This paper provides a study to model the transfer of NoV between food handlers and vegetables during salad preparation in school food services based on direct observation data. Three transfer pathways were modeled by considering different initial contamination sources (environment, handlers and contaminated produce). The probability of infection by NoV was also estimated based on the NoV levels at consumption obtained from each simulated transfer pathway. A scenario analysis ranging a wide concentration from 102 to 107 NoV infective particles was performed to represent different levels of NoV in the initial contamination sources. In addition, a sensitivity analysis was applied to identify the most important model inputs and determine the safest handling practices to be implemented in school food service operations. The pathway describing transfer from contaminated surfaces or handlers to foods indicated that initial levels of ≤104 NoV particles/fomite resulted in <0.5% cases per serving of NoV infection. When initial levels were higher, % cases of NoV infection was estimated to be ca. 3%. This rise in % cases of infection was linked to higher doses (5% serving with ≥15 NoV particles/serving) and prevalence levels at consumption (>0.2). In the pathway modeling cross contamination from contaminated vegetables to non-contaminated vegetables, all scenarios could lead to infected individuals, although number of cases of infection were lower (<1.3%), despite concentration levels were higher. On the contrary, for this pathway, prevalence was 2-fold lower than that observed in the pathways describing transfer from contaminated surfaces and hands. Based on the sensitivity analysis, NoV transfers to fresh produce may be minimized by improving hand washing, and therefore effective training programs need to be carried out specifically addressing hand washing. Moreover, the produce's washing step showed to be an effective control measure, depending on the desinfectant efficacy, by reducing % cases of NoV infection from 6 to 1%. The model in this study might be used, in the future, to evaluate the impact on the risk associated with NoV transmission of specific and effective training programs, aimed at food chain operators.

Reduction of fumonisin B1 in corn grits by single-screw extrusion.

This study was designed to determine the efficacy of extrusion in reducing fumonisin B1 in corn flaking grits in the presence and absence of glucose. In addition, degradation products of fumonisin B1 during extrusion were identified and quantitated with a mass balance approach. Uncontaminated clean corn grits, grits spiked with 30 microg/g fumonisin B1, and grits fermented with Fusarium verticillioides M-2552 (40-50 microg/g fumonisin B1) were extruded in the presence and absence of glucose (10%, w/w) using a single-screw extruder. Extrusion decreased fumonisin B1 by 21-37%, whereas the same process with added glucose further decreased fumonisin B1 by 77-87%. LC-fluorescence and LC-MS showed that most fumonisin in the extruded samples without added glucose was the fumonisin B1 form, whereas the main degradation product in grits extruded with glucose was N-(deoxy- d-fructos-1-yl)fumonisin B1. The formation of hydrolyzed fumonisin B1 was not significant during extrusion. Results suggest that extrusion in the presence of glucose may reduce fumonisin B1 in corn grits significantly.

Reduced toxicity of fumonisin B1 in corn grits by single-screw extrusion.

Corn grits spiked with 30 microg/g fumonisin B1 and two batches of grits fermented with Fusarium verticillioides (batch 1 contained 33 microg/g, and batch 2 contained 48 microg/g fumonisin B1), which were extruded by a single-screw extruder with and without glucose (10%, dry weight basis) supplementation were fed to rats. Control groups were fed uncontaminated grits. Extrusion with glucose more effectively reduced fumonisin B1 concentrations of the grits (75 to 85%) than did extrusion alone (10 to 28%). With one exception, the fumonisin B1-spiked and fermented extrusion products caused moderately severe kidney lesions and reduced kidney weights, effects typically found in fumonisin-exposed rats. Lesions in rats fed the least contaminated grits (batch 1) after extrusion with 10% glucose were, however, significantly less severe and not accompanied by kidney weight changes. Therefore, extrusion with glucose supplementation is potentially useful for safely reducing the toxicity of fumonisins in corn-based products and studies to determine the optimal conditions for its use are warranted.

Antibacterial effects of long-chain polyphosphates on selected spoilage and pathogenic bacteria.

The antimicrobial activities of four long-chain food-grade polyphosphates were studied at concentrations allowed in the food industry (<5,000 ppm) in defined basal media by determining the inhibition of growth of three gram-negative and four gram-positive spoilage and pathogenic bacteria. Both generation time and lag phase of Escherichia coli K-12, E. coli O157: H7, and Salmonella Typhimurium were increased with all of the polyphosphates tested. Bacillus subtilis and Staphylococcus aureus were more sensitive to polyphosphates, but not in all cases, with multiphased growth. The growth of Lactobacillus plantarum was inhibited by polyphosphates at concentrations above 750 ppm, but the lag time of Listeria monocytogenes was shortened by the presence of polyphosphates. No single polyphosphate was maximally inhibitory against all bacteria. Polyphosphates with chain lengths of 12 to 15 were significantly different from those with chain lengths of 18 to 21 depending on the organism and concentrations of polyphosphate used. Overall, higher polyphosphate concentrations resulted in greater inhibition of bacterial growth.

Renal toxicity through AhR, PXR, and Nrf2 signaling pathway activation of ochratoxin A-induced oxidative stress in kidney cells.

Because ochratoxin A (OTA) is widely found in foods, people are susceptible to OTA exposure. The mechanism leading to renal toxicity induced by OTA remains unclear. The aim of this study was to investigate OTA-induced toxicity in human proximal tubule HK-2 cells. OTA decreased cell viability, and the expression of kidney injury molecule-1 (KIM-1), a kidney damage marker, was increased when HK-2 cells were exposed to OTA. Additionally, OTA treatment of cells increased intracellular reactive oxygen species and malondialdehyde and decreased glutathione levels. OTA-treated cells induced the aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) genes followed by induction of the cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP3A4 genes representing phase I enzyme. The mRNA expression of phase II enzymes such as heme oxygenase-1, nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase 1, and glutamate cysteine ligase catalytic subunit were upregulated by activation of NF-E2-related factor 2 (Nrf2) translocation by treatment with OTA. The response of OTA-orally administered mice also showed marked increases in these enzymes as well as KIM-1. These results indicate that OTA induces phase I and II enzymes through the AhR, PXR, and Nrf2 signaling pathways in HK-2 cells, which may lead to modulation of proximal tubule injury.

Summary of the ACS Symposium on Public Health Perspectives of Mycotoxins in Food.

A symposium entitled "Public Health Perspectives of Mycotoxins in Food" was held at the 251st American Chemical Society (ACS) Meeting in March 2016 in San Diego, CA, and was sponsored by the ACS Division of Agricultural and Food Chemistry. The purpose of the symposium was to convene the leading mycotoxin researchers throughout the world to discuss the current state of knowledge as well as research needs with respect to evaluating the toxicological properties of mycotoxins and ways to detect, control, and reduce human and animal exposure to these natural toxins. A total of 23 presentations were delivered by speakers representing academic, government, and industrial institutions from North America, Europe, Asia, and Africa. The presentations covered such diverse topics as a historical perspective on the discovery of the major fungal toxins, occurrence of mycotoxins in food and feed, toxicological properties of mycotoxins and their influence on public health, analytical methods for mycotoxins, pre- and postharvest control of mycotoxins, and regulatory aspects. This paper is intended to provide a brief summary of the presentations as well as a record of the proceedings of the symposium.