Nontoxic Enantiomeric Reference Materials for Saxitoxins.

Saxitoxin (STX) is a potent neurotoxin that is biosynthesized by toxic dinoflagellates and accumulated in shellfish via the food chain. STX and its various analogues are now monitored in shellfish by the hygiene authorities in many countries with instrumental analytical methods, which require calibration with standards. Unfortunately, STX is registered as a chemical warfare agent in Schedule 1 of the Chemical Weapons Convention, and this has made it difficult to import calibration standards into some countries. We aimed to avoid violation of the Chemical Weapons Convention and facilitate analyses by preparing calibration standards based on unnatural nontoxic antipodal STXs (ent-STXs) with the same physicochemical properties as natural STXs. Our findings demonstrate that the nontoxic ent-STXs can be safely utilized as alternative reference materials of STXs in the routine monitoring program by the local authorities and consequently can lead to reduced usage of STX.

[Rapid Screening System for Paralytic Shellfish Toxins in Bivalves by Oligonucleotide Lateral Flow Immunoassay].

The mouse bioassay (MBA) for paralytic shellfish toxins (PSTs) in bivalves has been used as an official method in Japan. It is necessary to develop an alternative method to animal experiments in PSTs assay because 3Rs (Replacement, Reduction, and Refinement) of animal experiments are required from the animal welfare point of view. Various methods such as HPLC-FL, receptor binding assay, LC-MS/MS and ELISA have been established to detect PSTs without performing animal experiments. The present study was undertaken to develop a screening method using oligonucleotide lateral flow immunoassay (OLFIA) for detecting PSTs in bivalves. The screening level was defined as positive at 2 MU/g of MBA that is the half regulation limit of PSTs monitoring in Japan. All 20 positive (equal to or more than 2 MU/g) samples judged from MBA showed a positive reaction in the OLFIA. No positive samples resulted in a false negative reaction. The OLFIA exhibited high accuracy at 2 MU/g of screening criteria. The authors demonstrated here that the OLFIA can be useful for rapid detection of PSTs in bivalves.

Sodium Hypochlorite Pentahydrate as a Reagent for the Cleavage of trans-Cyclic Glycols.

Sodium hypochlorite pentahydrate (NaOCl·5H2O) can be used toward the efficient glycol cleavage of trans-cyclic glycols, which are generally resistant to this transformation. Interestingly, the reaction of cis-cyclic glycols with NaOCl·5H2O is slower than that observed for the corresponding trans-isomer. This trans selectivity is in sharp contrast to traditional oxidants used for glycol cleavage. Acyclic glycols can also react efficiently with NaOCl·5H2O to form their corresponding carbonyl compounds in high yield.

Correction to: Effects of periodontal treatment on carotid intima-media thickness in patients with lifestyle-related diseases: Japanese prospective multicentre observational study.

Unfortunately, in Table-5 of the original article, the parameter in the 5th row was published incorrectly as "LDL-C (mg/dL)". The correct parameter should read as "HDL-C (mg/dL)".

Effects of periodontal treatment on carotid intima-media thickness in patients with lifestyle-related diseases: Japanese prospective multicentre observational study.

Atherosclerosis, a chronic inflammatory disease in arterial blood vessels, is one of the major causes of death in worldwide. Meanwhile, periodontal disease is a chronic inflammatory disease caused by infection with periodontal pathogens such as P. gingivalis (Porphyromonas gingivalis). Several studies have reported association between periodontal infection and atherosclerosis, but direct investigation about the effects of periodontal treatment on atherosclerosis has not been reported. We have planned Japanese local clinics to determine the relationship between periodontal disease and atherosclerosis under collaborative with medical and dental care. A prospective, multicentre, observational study was conducted including 38 medical patients with lifestyle-related diseases in the stable period under consultation at participating medical clinics and 92 periodontal patients not undergoing medical treatment but who were consulting at participating dental clinics. Systemic and periodontal examinations were performed before and after periodontal treatment. At baseline, LDL-C (low-density lipoprotein cholesterol) levels and percentage (%) of mobile teeth were positively related to plasma IgG (immunoglobulin) antibody titer against P. gingivalis with multivariate analysis. Corresponding to improvements in periodontal clinical parameters after treatment, right and left max IMT (maximum intima-media thickness) levels were decreased significantly after treatment (SPT-S: start of supportive periodontal therapy, SPT-1y: at 1 year under SPT, and SPT-3y: at 3 years under SPT). The present study has clarified our previous univariate analysis results, wherein P. gingivalis infection was positively associated with progression of atherosclerosis. Thus, routine screening using plasma IgG antibody titer against P. gingivalis and periodontal treatment under collaborative with medical and dental care may prevent cardiovascular accidents caused by atherosclerosis.

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata.

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell(-1)·day(-1)) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (Rtox) was not affected by temperature. During EG, the average specific toxin production rate (µtox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day(-1) at 30 °C, and then decreased. Over the entire growth span, µtox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µtox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.

A convenient HPLC method for detection of okadaic acid analogs as 9-anthrylmethyl esters with automated sample cleanup by column switching.

A convenient HPLC-fluorometric detection (FLD) method for okadaic acid (OA) analogs as 9-anthrylmethyl esters was developed with the addition of column switching to simplify and automate cleanup. Methanol extracts of shellfish were first treated to hydrolyze OA esters and then reacted with 9-anthryldiazomethane (ADAM). ADAM derivatives of OA and dinophysistoxin-1 (DTX1) were subsequently determined by HPLC-FLD following automated column-switching cleanup. The LOD (S/N = 3) and LOQ (S/N = 10) of OA and DTX1 obtained from bivalves fortified with toxin in our method were approximately 2.6 and 8.6 ng/g whole meat, respectively. The recoveries of OA and DTX1 at all fortification levels of bivalve extracts ranged from 90 to 113%, with RSD values of 0.9-9.9%. The new method is applicable to the routine monitoring of OA analogs as an inexpensive and convenient alternative to HPLC/MS.

Risk factors affecting third molar autotransplantation during 5 and 10 years.

The aim of this study was to investigate risk factors affecting 5- and 10-year survival in autotransplantation of third molars with complete root formation at dental clinics. Participating dentists were requested to provide information on transplantations performed between 1 January 1990 and 31 December 2009. After data screening and elimination, 183 teeth in 171 men aged 20-72 years (mean, 44.8 years) and 205 teeth in 189 women aged 20-74 years (mean, 42.0 years) were included in the study. A single-factor analysis using the log-rank test revealed that the following factors had a significant influence (p<0.05) on 5-year survival in transplanted teeth in men: recipient site in the maxilla and fewer than 25 present teeth; those for 10-year survival, on the other hand, were recipient site tooth extraction due to periodontal disease, recipient site in the maxilla, fewer than 25 present teeth, and Eichner index Group B1 to C. Cox regression analysis revealed that the odds ratio for 5-year survival for recipient site in the maxilla was 2.873 (95% CI, 1.073-7.695), while that for 10-year survival was 3.713 (95% CI, 1.601-8.609) for recipient site extraction due to periodontal disease, 2.190 (95% CI, 1.021-4.700) for recipient site in the maxilla, and 3.110 (95% CI, 1.470-6.581) for fewer than 25 present teeth. In women, the log-rank test indicated experience of less than 10-year in performing treatment as a significant factor (p <0.05) in 5-year survival. These results suggest that medium-term survival in transplanted teeth is influenced by operational risk factors in women, while long-term survival in transplanted teeth is influenced by individual oral status in men.

A case of paralytic shellfish poisoning caused by consumption of visceral balls from geoduck Panopea japonica in Japan.

In the end of March 2018, an unprecedented food poisoning incident due to ingestion of the visceral balls of geoduck Panopea japonica occurred in Japan. The patient, presented with symptoms of numbness on the lips and general weakness, was diagnosed as paralytic shellfish poisoning (PSP). The patient immediately treated with the mechanical ventilation recovered and left the hospital after 3 days treatment. Saxitoxins (STXs) in the plasma and urinary samples collected from the patient on the first and second day after hospitalization were analyzed by ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) and liquid chromatography with post-column fluorescent detection (LC/FLD). The STXs levels of 499.1 and 6.0 µg/L of STX dihydrochloride equivalent (STX·2HCl eq.) were quantitated by LC/FLD in the urinary samples on the first and second day, respectively. In addition, geoducks harvested from the same areas of the PSP causative specimens after the incident were analyzed by LC/FLD, and the results showed the level of STXs in their whole bodies of the geoducks exceeding 0.8 mg STX·2HCl eq./kg which is the maximum levels of STX in CODEX STAN 292-2008. Prominent toxins in STXs that detected in urinary and geoduck samples and identified by UHPLC/MS/MS and LC/FLD were gonyautoxin-1+4 (GTX1+4). These results concluded that the incident was the food poisoning due to STXs accumulated in the geoducks. This is the first PSP case caused by consumption of geoducks in Japan. This is also the first PSP case that causative toxins are detected in urinary samples of patients involved in PSP in Japan.

New azaspiracid analogues detected as bi-charged ions in Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters.

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

Survival rate in autotransplanted premolars with complete root formation: a retrospective clinical survey.

The purpose of this study was to analyze the survival rate in autotransplanted premolars with complete root formation in dental clinics. Participating dentists were requested to provide information on transplantations they had undertaken between 1 January 1990 and 31 December 2010. Data on a total of 708 teeth from 637 patients were collected. Data for other tooth types and for teeth with incomplete root formation were eliminated. In this study, data on 40 teeth in 35 patients were analyzed. Participants consisted of 17 men and 18 women ranging from 24 to 79 years in age (mean age, 43.7 years). The cumulative survival rate was 100% at the 5-year mark and 72.7% at 10 years, as calculated by the Kaplan-Meier method. Single-factor analysis revealed that "transplanted to the molar regions" was a significant risk factor (p<0.05) influencing the survival of transplanted teeth. However, a Cox regression analysis showed no significance. The results of this study suggest that, in cases where there is a suitable donor tooth and the oral condition is good, premolar autotransplantation is a viable treatment option, even when there is complete root formation in the donor teeth.

Procedures for treating spaces vacated by loss of transplanted teeth.

The main reasons for loss of autotransplanted teeth are different from those involved in natural teeth loss. The aim of this study was to investigate which procedures were employed to treat spaces vacated when autotransplanted teeth were lost. Participating dentists were requested to provide information on transplantations they had undertaken. A total of 614 teeth in 552 patients (37 dentists) ranging in age from 17 to 79 years (mean age: 44.1 years) were examined. A total of 102 transplanted teeth were lost during the observation period. Procedures for treatment of spaces vacated were not influenced by main reason for transplanted tooth loss. The procedure used to treat depended on the original prosthodontic treatment of the transplanted teeth. For single crowns, the spaces were left empty (33.9%) or replaced by bridge work (30.5%), implants (20.3%), or dentures (10.2%). For single crowns in the upper and lower second molar regions, the spaces were usually left empty (upper 100%, lower 71.4%), while for those in the upper and lower first molar regions, the spaces were often replaced by bridge work (upper 41.7%, lower 50.0%). For bridge abutments, spaces were replaced by dentures (42.9%), implants (33.3%), or left empty (14.3%), and in the lower second molar region, they were mostly replaced by implants (5 cases, 41.7%). For most denture abutment cases, the spaces were replaced by dentures (88.9%). During the survival period of the transplanted teeth, the masticatory burden on the other teeth is reduced and the adjacent teeth are supported by the transplanted tooth. Even if transplanted teeth are eventually lost, traditional procedures can be performed to fill the vacated space.

Azaspiracid accumulation in Japanese coastal bivalves and ascidians fed with Azadinium poporum producing azaspiracid-2 as the dominant toxin component.

The filter-feeding bivalves often accumulate marine toxins by feeding on toxic dinoflagellates that produce marine toxins. Azaspiracids (AZAs) are a group of lipophilic polyether toxins which have been detected in a variety of organisms in many countries. In our present study, accumulation kinetics and toxin distributions in the tissues of seven bivalve species and ascidians relevant to Japanese coastal waters were investigated by experimentally feeding a toxic dinoflagellate Azadinium poporum, which produces azaspiracid-2 (AZA2) as the dominant toxin component. All bivalve species and ascidians investigated in this study had the capability to accumulate AZA2 and no metabolites of AZA2 were detected in the bivalves and the ascidians. Japanese short-neck clams, Japanese oysters, Pacific oysters and ascidians accumulated AZA2 with the highest concentrations on the hepatopancreas, whereas the highest concentrations of AZA2 were found on the gills in surf clams and horse clams. Hard clams and cockles accumulated high levels of AZA2 in both the hepatopancreas and the gills. As far as we know, this is the first report describing detailed tissue distribution of AZAs in several bivalve species other than mussels (M. edulis) and scallops (P. maximus). Variation of accumulation rates of AZA2 in Japanese short-neck clams on different cell densities or temperatures were observed.

Complex profiles of azaspiracid analogues in two culture strains of Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters determined by LC-MS/MS.

Lipophilic marine biotoxins azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters. In our present study, AZA analogues in two strains (mdd421 and HM536) of A. poporum were analyzed by several detection techniques on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). The dominant AZA analogue in the Japanese A. poporum strains was AZA2. Other known AZA analogues were AZA11, AZA35, AZA2 methyl ester and AZA2 phosphate ester. Besides these AZAs, thirteen new AZA analogues were discovered in the two strains. A putative AZA analogue (Compound 1) with the smallest molecular weight ever found in nature was also discovered in the two strains. This is the first report describing detailed AZA profiles in Japanese isolates of A. poporum.

Identification of Fish Species and Toxins Implicated in a Snapper Food Poisoning Event in Sabah, Malaysia, 2017.

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian countries, few have been confirmed by ciguatoxins identification, resulting in limited information for the correct diagnosis of this food-borne disease. In the present study, ciguatoxin-1B (CTX-1B) in red snapper (Lutjanus bohar) implicated in a CFP case in Sabah, Malaysia, in December 2017 was determined by single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Continuous consumption of the toxic fish likely resulted in CFP, even when the toxin concentration in the fish consumed was low. The identification of the fish species was performed using the molecular characterization of the mitochondrial cytochrome c oxidase subunit I gene marker, with a phylogenetic analysis of the genus Lutjanus. This is the first report identifying the causative toxin in fish-implicated CFP in Malaysia.

Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06.

Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

Quantitation of Tetrodotoxin and Its Analogues with a Combination of Liquid Chromatography-Tandem Mass Spectrometry and Quantitative 1H-NMR Spectroscopy.

Tetrodotoxin and its analogues are the causative toxins of pufferfish poisoning. Tetrodotoxin has been recently detected in bivalve mollusks collected in New Zealand and Europe, highlighting the need to include tetrodotoxin in monitoring programs for bivalves by instrumental methods. In the present study, tetrodotoxin and its analogues in commercially available tetrodotoxin reagents were quantitated accurately by quantitative 1H-nuclear magnetic resonance (qNMR) spectroscopy. The results were applied to estimate relative molar responses of tetrodotoxin and its analogues in hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC/MS/MS). All four components (tetrodotoxin hemilactal form (1), tetrodotoxin 10,7-lactone form (2), 4-epitetrodotoxin (3), and 4,9-anhydrotetrodotoxin (4)) generated by equilibrating tetrodotoxin in aqueous solution were prepared as a mixture. From the HSQC spectrum of the mixture, the separated signals derived from three components, excluding 1, were selected and used for the quantitation. In addition, the relative molar responses of 3 and 4 on HILIC/MS/MS were calculated to be 0.73 and 0.46, respectively. These values could be useful for quantitation of 3 and 4 using the tetrodotoxin standard by HILIC/MS/MS. Our results also indicate that qNMR is useful for preparation of tetrodotoxin certified reference material.

Development of Ultra-Performance Liquid Chromatography with Post-Column Fluorescent Derivatization for the Rapid Detection of Saxitoxin Analogues and Analysis of Bivalve Monitoring Samples.

Saxitoxin (STX) and its analogues produced by toxic dinoflagellates accumulate in bivalves, and routine monitoring of bivalves is important to prevent cases of human poisoning. In this study, we describe a rapid detection method for the analysis of STXs using ultra-performance liquid chromatography with post-column fluorescent detection and to investigate water depths and sampling points optimal for shellfish toxin monitoring. Cultured scallops (Mizuhopecten yessoensis) and mussels (Mytilus galloprovincialis) were collected from various water depths and sampling points were used in this study. Irrespective of bivalve species, toxin concentrations in bivalves were lower at deeper water depths. The toxin concentrations of bivalves did not differ greatly when bivalves were collected from the same bay. Although the levels of contamination of bivalves with STXs can depend on various environmental and geographical factors, our findings are useful for formulating a sampling protocol for the prevention of harvesting contaminated shellfish.

Large-scale Cultivation of Gymnodinium Catenatum for Paralytic Shellfish Poisoning Toxin Standards.

An adequate supply of standard reference material for paralytic shellfish toxins (PSTs) is critical for the accurate chemical quantification using high performance liquid chromatography (HPLC) with fluorescent detection, liquid chromatography-tandem mass spectrometry (LC-MS/MS), biological analysis of these toxins using enzyme-linked immunosorbent assay (ELISA), and immunochromatography. Large batch cultivation for the chain forming species G. catenatum, producers of PSTs of N-sulfocarbamoyl-11-hydroxysulfate toxins (C1 and C2), gonyautoxin 5 (GTX5) and gonyautoxin 6 (GTX6), was investigated using 10 L round-bottom flasks with aeration for the production of GTX5 and GTX6. Aeration rates of 200 mL/min and 500 mL/min were compared, demonstrating that the 500 mL/min aeration rate was adequate to eliminate aggregation of cells. The highest cell density of G. catenatum in 500 mL/min aeration treatment was 9,878 ± 2,617 cells/ml on day 28. Total toxin yield during 10 L cultivation with 500 mL/min aeration was calculated at 30.9 ± 3.6 µmol on day 25, with GTX5 and GTX6 calculated at 3.9 ± 0.7 µmol and 11.4 ± 1.4 µmol, respectively. This simple aeration method will contribute to the more efficient production of PST reference materials.

Anatomical Distribution of Diarrhetic Shellfish Toxins (DSTs) in the Japanese Scallop Patinopecten yessoensis and Individual Variability in Scallops and Mytilus edulis Mussels: Statistical Considerations.

Diarrhetic shellfish toxins (DSTs) are a group of phycotoxins that include okadaic acid (OA)/dinophysistoxin (DTX) analogues. At present, detailed data on the distribution of DST is insufficient, and studies of the appropriate sample sizes are lacking. This study investigated the DST frequency distribution in scallops and mussels by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and a resampling analysis of existing data was carried out. The DST population-interval and the necessary sample size were also estimated. DSTs are localized in the scallop digestive-gland, and the DST concentrations in scallops were water-depth-dependent. DST concentrations in scallops and mussels showed normal distributions, but mussels tended to contain more DSTs than scallops. In the statistical resampling analysis of the acquired data on scallops and mussels, especially that using the bootstrap method, sample size was difficult to estimate when the DST variation was large. Although the DST population-interval could be statistically estimated from the sample standard deviation of three samples, the sample size corresponded to the risk management level, and the use of 13 or more samples was preferable. The statistical methods used here to analyze individual contents and estimate population content-intervals could be applied in various situations and for shellfish toxins other than DSTs.