Exploring the characteristics of Burkholderia gladioli pathovar cocovenenans: Growth, bongkrekic acid production, and potential risks of food contamination in wet rice noodles and vermicelli.

This research investigated the presence of Burkholderia gladioli pathovar cocovenenans (BGC) in wet rice and starch products, Tremella, and Auricularia auricula in Guangzhou, China. It examined BGC growth and bongkrekic acid (BA) production in wet rice noodles and vermicelli with varying rice flour, edible starch ratios, and oil concentrations. A qualitative analysis of 482 samples revealed a detection rate of 0.62%, with three positive for BGC. Rice flour-based wet rice noodles had BA concentrations of 13.67 ± 0.64 mg/kg, 2.92 times higher than 100% corn starch samples (4.68 ± 0.54 mg/kg). Wet rice noodles with 4% soybean oil had a BA concentration of 31.72 ± 9.41 mg/kg, 5.74 times higher than those without soybean oil (5.53 ± 1.23 mg/kg). The BA concentration correlated positively (r = 0.707, P < 0.05) with BGC contamination levels. Low temperatures (4 °C and -18 °C) inhibited BGC growth and BA production, while higher storage temperatures (26 °C and 32 °C) promoted BGC proliferation and increased BA production. Reducing edible oil use and increasing edible starch can mitigate the risk of BGC-related food poisoning in wet rice noodles and vermicelli production. Further research is needed to find alternative oils that do not enhance BA production. Strengthening prevention and control measures is crucial across the entire production chain to address BGC contamination and BA production.

The toxicokinetic and extracorporeal removal of bongkrekic acid during blood purification therapies: A case report.

Bongkrekic acid (BA) poisoning can progress rapidly and lead to the failure of multiple organs, such as brain, liver and kidney. The mortality of BA poisoning is 40-100%. Little information is available on the toxicokinetic parameters of BA in human. Although hemodialysis is widely utilized for patients with severe BA poisoning, the exact amount of BA removed by hemodialysis is poorly documented. We analyzed toxicokinetic parameters, endogenous clearance and hemodialysis clearance in a patient with BA poisoning. A 27-year-old male developed symptoms of severe diarrhea, nausea, vomiting and weakness after eating rice noodles for more than one day. The patient developed multiple organ failures, especially the liver. Initial serum BA concentration was 0.5µg/mL. He received plasmapheresis, routing, and Oxiris-based Continuous Renal Replacement Therapy (CRRT). The whole blood, serum, urine and dialysate BA concentrations were collected and analyzed hourly. Toxicokinetic parameters relationships were determined using noncompartmental analysis. The clearances were determined using standard pharmacokinetic calculations. The disposition of BA was characterized by a long half-life (t1/2 of 102) and high max plasma (CL of 129,000 L/h/kg) following ingestion of contaminated food. The average serum clearance of BA during PE is remarkable higher than CRRT and the endogenous clearance. In contrast, the rates of decline in blood levels during the CRRT treatments were similar to the natural rate of decline. The total amount of BA removed by Plasmapheresis was 5.51mg. However, most CRRT failed to eliminate BA. We report a rare case of BA poisoning with a complication of liver failure and acute kidney damage. The patient expired, even with supportive care, plasmapheresis and hemodialysis. Analysis of whole blood, serum, urine and dialysate concentrations showed limited efficacy of CRRT in removing BA from blood. In contrast, there was significant extraction of BA from Plasmapheresis.

Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

Pan-genome analysis of the Burkholderia gladioli PV. Cocovenenans reveal the extent of variation in the toxigenic gene cluster.

Burkholderia gladioli has been reported as the pathogen responsible for cases of foodborne illness in many countries. The poisonous bongkrekic acid (BA) produced by B. gladioli was linked to a gene cluster absent in non-pathogenic strains. The whole genome sequence of eight bacteria strains, which were screened from the collected 175 raw food and environmental samples, were assembled and analyzed to detect a significant association of 19 protein-coding genes with the pathogenic status. Except for the common BA synthesis-related gene, several other genes, including the toxin-antitoxin genes, were also absent in the non-pathogenic strains. The bacteria strains with the BA gene cluster were found to form a single cluster in the analysis of all B. gladioli genome assemblies for the variants in the gene cluster. Divergence of this cluster was detected in the analysis for both the flanking sequences and those of the whole genome level, which indicates its complex origin. Genome recombination was found to cause a precise sequence deletion in the gene cluster region, which was found to be predominant in the non-pathogenic strains indicating the possible effect of horizontal gene transfer. Our study provided new information and resources for understanding the evolution and divergence of the B. gladioli species.

Molecular docking and molecular dynamics simulation study on the toxicity mechanism of bongkrekic acid.

BKA belongs to gram-negative brevibacterium. It can cause poisoning in humans or animals and can be fatal in severe cases. There are few investigations on toxic mechanisms of BKA because of foodborne factors. MD simulations were used to study the stability and intermolecular interactions of BKA and ANT complexes to reveal the mechanism of BKA in this paper. BKA blocked ANT protein translocation mainly through Van der Waals force, hydrophobic and hydrogen bonding interactions by the MD simulations. The conformational flexibility of the complex system during different simulation times indicated that BKA affected the conformational changes of ANT through strong interactions of hydrogen bonds with active domain residues Gln-93, Tyr-196, Arg-287 and Arg-245. The results of binding free energy, principal component analysis, hydrophobic interactions and root-mean-square fluctuation showed that the prominent binding force of Tyr-196 with C26 of BKA was significant to the toxicity. The active site interactions analysis indicated that the essential positively charged polar amino acids which play a crucial role within the active site of the ANT protein undergo conformational changes with BKA as the branch point.

Epidemiology of foodborne bongkrekic acid poisoning outbreaks in China, 2010 to 2020.

Foodborne bongkrekic acid (BA) poisoning is a fatal foodborne disease in China. From 2010-2020, a total of 19 BA poisoning outbreaks were reported to the China National Foodborne Disease Outbreak Surveillance System. These outbreaks involved 146 illnesses, 139 hospitalizations, and 43 deaths, with a case-fatality rate of 29.5%. Approximately 73.3% of the outbreaks occurred in South and Southwest China. Homemade fermented corn flour products, tremella, and sweet potato flour and corn flour products (jelly) caused more early outbreaks, and novel vehicles (wet rice noodles and Auricularia auricula) were associated with later outbreaks in the study period. Outbreaks most frequently occurred at home (79.0%) and in restaurants (21.0%). The prohibition of traditional processed homemade fermented corn flour products and improvement in bongkrekic acid poisoning case identification and early treatment have resulted in a reduction in the case-fatality rate.

KH-17, a simplified derivative of bongkrekic acid, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane.

Two natural products, bongkrekic acid and carboxyatractyloside, are known to specifically inhibit the mitochondrial ADP/ATP carrier from its matrix side and cytosolic side, respectively, in concentration ranges of 10-6  M. In the present study, we investigated the manner of action of a synthetic bongkrekic acid derivative, KH-17, lacking three methyl groups, one methoxy group, and five internal double bonds, on the mitochondrial ADP/ATP carrier. At slightly acidic pH, KH-17 inhibited mitochondrial [3 H]ADP uptake, but its inhibitory action was about 10 times weaker than that of its parental compound, bongkrekic acid. The main site of action of KH-17 was confirmed as the matrix side of the ADP/ATP carrier by experiments using submitochondrial particles, which have an inside-out orientation of the inner mitochondrial membrane. However, when we added KH-17 to mitochondria at neutral pH, it had a weak inhibitory effect on [3 H]ADP uptake, and its inhibitory strength was similar to that of bongkrekic acid. These results indicated that KH-17 weakly inhibits the ADP/ATP carrier not only from the matrix side but also from the cytosolic side. To ascertain whether this interpretation was correct, we examined the effects of KH-17 and carboxyatractyloside on mitochondrial [3 H]ADP uptake at two [3 H]ADP concentrations. We found that both KH-17 and carboxyatractyloside showed a stronger inhibitory effect at the lower [3 H]ADP concentration. Therefore, we concluded that the bongkrekic acid derivative, KH-17, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane. These results suggested that the elimination of three methyl groups, one methoxy group, and five internal double bonds present in bongkrekic acid altered its manner of action towards the mitochondrial ADP/ATP carrier. Our data will help to improve our understanding of the interaction between bongkrekic acid and the mitochondrial ADP/ATP carrier.

Development and validation of a multianalyte method for quantification of aflatoxins and bongkrekic acid in rice and noodle products using PRiME-UHPLC-MS/MS method.

An analytical method based on PRiME (process, robustness, improvements, matrix effects, ease of use) HLB purification followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection has been developed for the determination aflatoxin B1, B2, G1 and G2 and bongkrekic acid in rice and noodle products. Five toxins were separated on a Waters BEH C18 column by gradient elution, scanned by ESI+ and ESI- dynamic switching and detected with MRM mode. LOD, LOQ, matrix effects, accuracy and precision of the developed method were investigated. Under the optimal sample pretreatment conditions, high sensitivity (LOQs: 0.20-0.40 µg/kg), good recoveries (80.5%-106.6%) and acceptable precision (2.4%-7.2%) were obtained for the analysis of the four aflatoxins and bongkrekic acid. This method was successfully applied to the analysis of rice and noodle products, demonstrating its applicability and suitability for the routine analysis of aflatoxins and bongkrekic acid in rice and noodle products.

[Fast determination of bongkrekic acid in plasma by high performance liquid chromatography-tandem mass spectrometry].

Objective: To establish a method for rapid determination of bongkrekic acid (BA) in plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods: In November 2020, plasma samples were extracted by methanol and acetonitrile (1∶1) and purified directly. The samples were separated by C18 column. Gradient elution was carried out with 5 mmol/L ammonium acetate water acetonitrile solution as mobile phase. Under the optimized instrument conditions, the electrospray ionization multiple reaction monitoring (MRM) mode was used, and the external standard method was used for quantitative analysis. Results: The linear relationship of BA in plasma was good in the concentration range of 2-100 µg/L, the correlation coefficient was 0.9998, the average recovery was 83.7%-112.0%, the relative standard deviation within and between batches was less than 10%, the detection limit of the method was 0.7 µg/L and the lower limit of quantification was 2.0 µg/L. Conclusion: The method is simple, rapid, accurate and sensitive, and can meet the requirements for the determination of BA in blood samples of poisoning patients.

Fast determination of bongkrekic acid in plasma by high performance liquid chromatography-tandem mass spectrometry / 中华劳动卫生职业病杂志

Objective: To establish a method for rapid determination of bongkrekic acid (BA) in plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods: In November 2020, plasma samples were extracted by methanol and acetonitrile (1∶1) and purified directly. The samples were separated by C18 column. Gradient elution was carried out with 5 mmol/L ammonium acetate water acetonitrile solution as mobile phase. Under the optimized instrument conditions, the electrospray ionization multiple reaction monitoring (MRM) mode was used, and the external standard method was used for quantitative analysis. Results: The linear relationship of BA in plasma was good in the concentration range of 2-100 μg/L, the correlation coefficient was 0.9998, the average recovery was 83.7%-112.0%, the relative standard deviation within and between batches was less than 10%, the detection limit of the method was 0.7 μg/L and the lower limit of quantification was 2.0 μg/L. Conclusion: The method is simple, rapid, accurate and sensitive, and can meet the requirements for the determination of BA in blood samples of poisoning patients.

[Determination of bongkrekic acid in tremella and auricularia auricular by improved QuEChERS method combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry].

An improved QuEChERS (quick, easy, cheap, effective, rugged, safe) method, combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was used to determine bongkrekic acid (BA) in tremella and auricularia auricular. BA is a fat-soluble, fatal bacterial toxin produced by the aerobic gram-negative bacteria Burkholderia gladioli pathovar cocovenenans. Tremella and auricularia auricular, which have rich nutritional values, are traditional edible fungi in China that are very popular among Chinese consumers. However, tremella and auricularia auricular are easily contaminated by BA during storage and foaming, and this poses a great threat to food safety and human health. Therefore, establishing a rapid and efficient analysis method for detecting BA in tremella and auricularia auricular is of considerable significance. However, the BA concentration in the actual sample is very low, and the matrices of tremella fuciformis and auricularia auricular are very complex. Thus, it is necessary to employ appropriate sample pretreatment technology to extract and purify BA from tremella and auricularia auricular samples prior to instrumental analysis. In this study, the QuEChERS method, combined with UHPLC-MS/MS, was used to detect BA in tremella and auricularia auricular. The key parameters, such as extraction solvent, extraction method, and adsorbent used for cleanup, were optimized to obtain high extraction efficiency. The content of acetic acid in the extraction solution strongly influenced the extraction efficiency of BA, and acetonitrile with 5%(v/v) acetic acid was determined to be the optimum extraction solvent. After salting out, the acetonitrile extract was purified by dispersive solid phase extraction using 200 mg C18 as a cleanup adsorbent. The sample was then separated on a Waters HSS T3 column (100 mm×2.1 mm, 1.8 µm), using a water solution containing 0.01% (v/v) formic acid and 0.05% (v/v) ammonia and methanol as mobile phases. MS analysis was performed using an electrospray ionization source in the negative and multiple reaction monitoring (MRM) modes. Under the optimized conditions, the matrix effects of UHPLC-MS/MS in tremella and auricularia auricular were -6.3% and -11.5%, respectively; this indicated that the method had a significant purification effect, and the sample matrix did not affect the MS detection of BA. Further study showed that in the concentration range of 1-200 µg/L, the square of the regression coefficient of the linear equation (R2) was greater than 0.999. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.15 µg/kg and 0.5 µg/kg, respectively. The average recoveries in samples spiked with 0.5, 10, and 50 µg/kg BA in tremella ranged from 92.4% to 102.6%, and the intra-day and inter-day relative standard deviations (RSDs) were 4.3%-4.9% and 3.2%-3.5%, respectively. For auricularia auricular, the average recoveries ranged from 89.6% to 102.3%, and the intra-day and inter-day RSDs were 2.4%-9.5% and 3.6%-4.1%, respectively. These results indicate that the proposed method has satisfactory sensitivity, accuracy, and precision. Finally, the method showed good performance when applied to the analysis of real samples. Compared with other reported methods, the LOD and LOQ of our proposed method were lower, with satisfactory recovery and precision. Taken together, this study provides an effective detection technology for the monitoring and risk control of BA in tremella and auricularia auricular.

Bongkrekic acid induced neutrophil extracellular traps via p38, ERK, PAD4, and P2X1-mediated signaling.

Bongkrekic acid (BKA) produced by pseudomonas cocovenenans is a deadly toxin, and is mainly found in spoiled or fermented foods. However, less is known on its immunotoxicity. Neutrophil extracellular traps (NETs) are a novel effector mechanism of neutrophils against invading pathogens, but excessive NETs also contribute to tissue damage. This study aimed to investigate NET formation triggered by BKA in murine neutrophils, and describe its characteristics and potential mechanisms. Our results showed that BKA triggered NET formation via co-localization of DNA and histone or MPO by immunostaining. Moreover, BKA-triggered NET formation was dose- and time-dependent via NET quantification based on Picogreen-derived fluorescence intensities. Furthermore, BKA increased ROS production in neutrophils. Pharmacological inhibition indicated that BKA-triggered NET formation was associated with ROS-p38 and -ERK signaling pathways, but independent on NADPH oxidase. Besides, PAD4 and P2X1 receptor also mediated BKA-triggered NET formation. To our knowledge, all these findings provide for the first time an initial understanding of BKA on innate immunity, which might be helpful for further investigation on BKA immunotoxicity.

Kill and cure: genomic phylogeny and bioactivity of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Determination of bongkrekic acid and isobongkrekic acid in rice noodles by HPLC-Orbitrap HRMS technology using magnetic halloysite nanotubes.

The existing extraction and detection methods of bongkrekic acid (BKA) and isobongkrekic acid (IBKA) are complex, time-consuming and solvent-consuming. In this work, a simple and fast pre-concentration procedure based on Fe3O4/HNTs was developed for the determination of BKA and IBKA in rice noodles using HPLC-Orbitrap HRMS. The structure and morphology of Fe3O4/HNTs was characterized by means of XRD, SEM, FT-IR and VSM. Parameters affecting the extraction efficiency including adsorbent amount, pH, extraction time, type and volume of eluent were investigated by employing the response surface method. Results indicated that the proposed method had favorable linearity in the concentration range of 2-200 µg/L with a correlation coefficient >0.998. Method LOD and LOQ were 0.3 µg/kg and 1.0 µg/kg, respectively. Finally, the method was successfully applied to determine BKA and IBKA in rice noodle samples from southern China with recoveries ranging from 79.8% to 102.6% and relative standard deviation (RSD) of 4.2%-7.1%.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

[Rapid determination of bongkrekic acid in Liushenqu by ultra performance liquid chromatography-tandem mass spectrometry].

A method for the rapid determination of biotoxin (bongkrekic acid) in the Liushenqu was established using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted with methanol by ultrasonication, then the pH was adjusted to 8 using ammonia. After filtration, the extract was purified using an Oasis Max strong anion exchange resin column. The Waters HSS T3 column (100 mm×2.1 mm, 1.8 µm) was used for the UPLC-MS/MS analysis. The mobile phase was acetonitrile-10 mmol/L ammonium formate solution (containing 0.1% (v/v) formic acid). MS analysis was performed using an electrospray ionization (ESI) source in the negative and multiple reaction monitoring (MRM) modes. Under the optimum conditions, the linear range of bongkrekic acid was 0.5-100 µg/L (correlations coefficient (R2)>0.99). The recoveries of the bongkrekic acid were 80.6%-85.3%. The intra-day and inter-day relative standard deviations (RSDs) were in the range of 4.2%-6.8% and 8.2%-13.2%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were 0.4 µg/kg and 1.2 µg/kg, respectively. The results showed that bongkrekic acid residues were detected in Liushenqu, thereby confirming the supporting role of our method for the risk monitoring of biotoxins in health foods and Chinese herbal medicines. This method is simple, easy, sensitive, and suitable for the determination of the bongkrekic acid residues in Liushenqu.

An Investigation of Bongkrekic Acid Poisoning Caused by Consumption of a Nonfermented Rice Noodle Product without Noticeable Signs of Spoilage.

Bongkrekic acid (BKA) is a tricarboxylic fatty acid that inhibits adenine nucleotide translocase as a kind of mitochondrial toxins. BKA is produced by the bacterium Burkholderia gladioli pathovar cocovenenans. An investigation was performed to determine the source of possible BKA poisoning of a family in H City, Guangdong Province, People's Republic of China, who consumed a commercially produced rice noodle product that was not fermented or noticeably spoiled. Clinical and food samples were tested. BKA concentration was detected by liquid chromatography-tandem mass spectrometry. We isolated and identified the suspicious strains from the rice noodles and performed toxicity determination through an animal experiment. BKA detected in the cases and the dead dog was 2.15 to about 343 µg/kg. The cases and dead dog shared a unique history of food exposure. The BKA in the factory's food samples was 150 and 160 µg/kg. All mice given the BKA extract by gavage died within 24 h. In conclusion, the food poisoning was caused by the high BKA concentration of expired (4 days over the 24-h shelf life) wet rice noodle products, with corn and wheat starch contaminated by B. gladioli cocovenenans. Different from traditional BKA poisoning caused by fermented and spoiled corn or coconut products, there was no noticeable spoilage because of the nonfermentation process and overused sodium dehydroacetate. The risk of BKA in wet rice noodle products and application of antiseptics, such as sodium dehydroacetate, in such food should be quantitatively evaluated to prevent the recurrence of similar events.

Bongkrekic acid poisoning: Severe liver function damage combined with multiple organ failure caused by eating spoiled food.

Bongkrekic acid (BA) poisoning can be caused by eating spoiled or fermented foods contaminated with pseudomonas cocovenenans. Although some in vitro studies have been reported on the use of purified BA to interfere with cell metabolism, few clinical or pathological data of BA poisoning on human due to food-borne factors are available for forensic appraisal. For the first time, we retrospectively report five cases of food-borne poisoning caused by eating rice noodles, a popular traditional food in Guangdong, China, and three of the victims died. All five victims were hospitalized with gastrointestinal symptoms such as nausea, vomiting and diarrhea and were treated with admission diagnosis of liver failure and acute kidney damage. Certain concentrations of BA were detected in the victims' peripheral blood serums at the hospitalization (ranging from 70-345 µg/L) and the suspected poisonous foods (0-810 ng/g) with LC-MS/MS technique. The results of forensic pathological examination showed that all three deceased had severe liver and kidney damage, accompanied by multiple organ congestion and edema, which were consistent with clinical diagnosis. Combined with the clinical records, we found that the difference in blood glucose between the deceased and survivors of the five victims may be an indication of the severity of the disease. In addition, we compared BA poisoning with other diseases that can cause acute liver function damage in terms of pathological characteristics and clinical manifestations, which has important reference significance for the diagnosis and forensic appraisal of this food-borne poisoning.

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.

Mitochondrial dysfunction induced by leflunomide and its active metabolite.

Leflunomide, an anti-inflammatory drug used for the treatment of rheumatoid arthritis, has been marked with a black box warning regarding an increased risk of liver injury. The active metabolite of leflunomide, A771726, which also carries a boxed warning about potential hepatotoxicity, has been marketed as teriflunomide for the treatment of relapsing multiple sclerosis. Thus far, however, the mechanism of liver injury associated with the two drugs has remained elusive. In this study, cytotoxicity assays showed that ATP depletion and subsequent LDH release were induced in a time- and concentration-dependent manner by leflunomide in HepG2 cells, and to a lesser extent, by A77 1726. The decline of cellular ATP levels caused by leflunomide was dramatically exacerbated when galactose was substituted for glucose as the sugar source, indicating a potential mitochondrial liability of leflunomide. By measuring the activities of immuno-captured mitochondrial oxidative phosphorylation (OXPHOS) complexes, we found that leflunomide and A77 1726 preferentially targeted complex V (F1FO ATP synthase), with IC50 values of 35.0 and 63.7 µM, respectively. Bongkrekic acid, a mitochondrial permeability transition pore blocker that targets adenine nucleotide translocase, profoundly attenuated mitochondrial membrane depolarization, ATP depletion, and LDH leakage induced by leflunomide and A77 1726. Substantial alterations of mitochondrial function at the transcript level were observed in leflunomide-treated HepG2 cells, whereas the effects of A77 1726 on the cellular transcriptome were much less profound. Our results suggest that mitochondrial dysfunction may be implicated in the hepatotoxicity associated with leflunomide and A77 1726, with the former exhibiting higher toxicity potency.