[This corrects the article DOI: 10.1371/journal.pone.0003364.].

Selenoneine (SEN), a selenium analog of ergothioneine (EGT), is widely distributed in marine fish and is a strong radical scavenger. Electron spin resonance spectrometry showed that SEN monomer and dimer directly scavenged ·OH generated by irradiating hydrogen peroxide (H2O2) with ultraviolet light. The radical scavenging capacity was stronger for SEN monomer, dimer, and EGT in that order. Mass spectrometry analyses revealed that the monomer and dimer were oxidized to SEN seleninic acid (SEN-seleninic acid) in the presence of H2O2, and that SEN-seleninic acid was reduced to SEN monomer by reduced glutathione (GSH). These reactions proceeded at physiological concentrations of H2O2 and GSH. Our findings suggest that SEN scavenges ·OH directly by a rapid, repetitive nonenzymatic reaction via self-oxidation and by its reduction back to SEN.

Identifying the sensory properties of fish consumers is important for providing their preferred seafood. Odor, flavor, texture, and appearance affected the sensory preference for fish. Moreover, several compounds are involved in sensory preference. This study focused on lipids that affect preference for seafood and examined the lipid content preference in marlin Kajikia audax sashimi. First, lipid contents and fatty acid compositions of different individual sizes and body parts of marlin were compared. Next, the preference for overall, taste, texture, odor, aftertaste, and lipid content in marlin sashimi taken from different parts of the body with different amounts of lipids was investigated using the paired preference method. Then the relationship of lipid content preference with other preferences were also evaluated using the Chi-square test. The crude lipid of big-sized marlin (51.1–55.1 kg) was 9.5%−13.2%, and the crude lipid of small-sized marlin (23.5–30.0 kg) was 0.8%−1.2%. The dorsal parts of big-sized marlin had lipid contents in the anterior parts higher than those in the posterior parts (p < 0.05). Whereas no differences were observed in the fatty acid composition of different body parts. The lipid content preference for the anterior dorsal part (high lipid part) was higher than that for the posterior dorsal part (lower lipid part) (p < 0.05). However, no difference in overall preference was observed. Additionally, the relationship between lipid content preference and other preference were indicated that the high lipid content parts preferred group significantly preferred the high lipid content parts at overall and taste evaluation (p < 0.01). On the other hand, the low lipid content parts preferred groups showed opposite evaluation. Therefore, the lipid content was associated with the preference for marlin sashimi and be classified into two groups: one prefers high lipid sashimi and the other prefers low lipid sashimi.

Oral intake of purified selenoneine and seafoods has been reported to result in selenoneine accumulation in erythrocytes in mice and human. In addition, Se-methylselenoneine was suggested to be produced as a metabolite of selenoneine in the urine and whole blood of humans. In order to confirm the molecular mechanism of production of Se-methylselenoneine, a stable isotope (Se-76) labeled selenoneine was biosynthesized using genetically modified fission yeast and administered to mice. The Se-76-labeled Se-methylselenoneine was detected in urine but Se-78 and Se-80-labeled Se-methylselenoneine arising from natural isotopes of Se was hardly detected. These results suggest that Se-methylselenoneine was a metabolite and the excreted form of selenoneine. The methylation of selenoneine in mice administered selenoneine continuously was evaluated by the analyses of organs using an online liquid chromatograph system with an inductively coupled plasma mass spectrometer (LC-ICP-MS). These experiments indicate that selenoneine is methylated in the liver and (or) kidneys.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 x 10(-8); exome-wide threshold: 2.5 x 10(-6)). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 x 10(-13)) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.

Selenoneine is a selenium-containing compound that exhibits strong radical-scavenging activity. Here we present a novel function of selenium in which selenoneine exhibits inhibitory activity against angiotensin-converting enzyme (ACE). ACE activity was strongly inhibited by selenoneine as compared to a typical peptide that decreases blood pressure in vitro. Kinetics analysis suggested that selenoneine inhibits ACE by competitive inhibition via the binding of selenoneine to zinc in the active center of ACE.

The novel organic selenium compound, selenoneine, is found in the blood of tuna and has metal-binding activity. In this report, selenoneine displays tyrosinase inhibitory activity. When murine B16 melanoma cells were cultured in the presence of 1.0 μM selenoneine, the melanin content in the cells was reduced to 46.5% compared with the cell-induced melanin synthesis, and cellular tyrosinase activity was suppressed. In 3D-cultured human melanocytes, melanin accumulation was also decreased, to 39.7% and 23.0% by 1.0 and 5.0 μM selenoneine, respectively, compared with the control cells. Both cellular and purified enzyme assays showed that selenoneine inhibited tyrosinase activity against the substrate, l-3,4-dihydroxyphenylalanine (l-DOPA). An in silico docking simulation study supported a molecular mechanism in which selenoneine chelates copper ions in the active center of tyrosinase and prevents the reaction between tyrosinase and l-DOPA. These findings suggest that selenoneine has a novel biological function by inhibiting tyrosinase via copper chelation.

We have monoclonal antibodies (mAbs) against CD4-1 (6D1) and CD8α (2C3) in ginbuna crucian carp Carassius auratus langsdorfii. In our previous studies we showed that 2C3 mAb positive cells are the primary cell type showing specific cytotoxicity against allogeneic targets, suggesting that CD8α+ lymphocytes in ginbuna are equivalent to cytotoxic T lymphocytes (CTLs) in mammals. We further demonstrated the helper T cell function of 6D1 mAb positive cells by studying mixed leukocyte culture (MLC) and hapten/carrier effects. Here, we report that our mAbs cross-react with zebrafish lymphocytes. First, mAbs 6D1 and 2C3 recognized 7-11% of zebrafish lymphocytes that were ZAP-70 positive and had the typical morphology of lymphocytes. Second, to verify the cell types reacting with the 6D1 and 2C3 mAbs we examined the expression profiles of zebrafish lymphocyte surface markers in FACS-sorted lymphocytes from kidney. cd4-1 (cd8a) and tcrac but not iglc transcripts were detected in 6D1(2C3)+ lymphocytes, whereas cd4-1 (cd8a) transcripts were not found in 6D1 (2C3)- lymphocytes. Third, we further confirmed that 6D1 reacted with zebrafish CD4-1 but not CD4-2, and 2C3 recognized zebrafish CD8α expressed on HEK293T cells. Collectively, these findings suggest that 6D1+ and 2C3+ lymphocytes in zebrafish are equivalent to CD4+ and CD8α+ T lymphocytes in mammals, respectively. Furthermore, we found the cross-reactivity of our 6D1 and 2C3 mAbs with other cyprinid species including goldfish, common carp and grass carp.

The relationship between freshness and odor components of the muscle of blue shark Prionace glauca caught by a longline fishing vessel and landed at Kesennuma fishing port was investigated. While the K value of the shark meat increased during ice storage in the fish hold on the vessel after catch, it scarcely increased during exposure to the open air for 6 hours after being landed. Trimethylamine and dimethylamine contents were scarcely detected in the shark muscle extract, so it was considered that they were not major factors of the odor. On the other hand, a small amount of ammonium content was detected in the shark muscle extract. As the ice-storage period in the fish hold on the vessel increased, the variation in the ammonium content increased. However, it hardly increased during exposure to the open air for 6 hours after being landed. Therefore, we considered the freshness of blue shark caught in the latter period of the voyage to be higher than that of those caught in the former period, and the intensity of the odor of blue shark caught in the latter period of the voyage to be lower than that of those caught in the former period.

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRN). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.

Coffin-Siris syndrome (CSS) is a congenital disorder characterized by growth deficiency, intellectual disability, microcephaly, characteristic facial features and hypoplastic nails of the fifth fingers and/or toes. We previously identified mutations in five genes encoding subunits of the BAF complex, in 55% of CSS patients. Here we perform whole-exome sequencing in additional CSS patients, identifying de novo SOX11 mutations in two patients with a mild CSS phenotype. sox11a/b knockdown in zebrafish causes brain abnormalities, potentially explaining the brain phenotype of CSS. SOX11 is the downstream transcriptional factor of the PAX6-BAF complex, highlighting the importance of the BAF complex and SOX11 transcriptional network in brain development.

Nucleotide sequences in internal transcribed spacer (ITS)-1 region derived from dried nori products produced in Japan, China, and the Republic of Korea were compared. Thalli contained in the Japanese products were genetically homogenous, and their nucleotide sequences in ITS-1 were identical to those of the reference strains of Pyropia yezoensis f. narawaensis. In Chinese products, the thalli were related to P. yezoensis strain Minomiasakusa. In contrast, the thalli in the Korean products were genetically heterogeneous, and several different P. yezoensis strains and other Pyropia spp. were used for dried nori products. In some thalli produced in both China and Korea, the DNA sequences of the ITS-1 region were identical with that of Japan, suggesting that the cultivar strains might have been transplanted from Japan to China in recent years. The 432-bp-long nucleotide sequences in the ITS-1 region of thalli derived from Japanese origin were cleaved to two restriction fragments at 154 and 278 bp by cleavage of PCR-amplified products using MspI. Conversely, almost all of the corresponding sequences derived from China and Korea were lacking MspI or other restriction patterns, except for nori products from some areas that cultivate a closely related strain to the Japanese cultivar.

Selenium‐containing imidazole compound, 2‐selenyl‐Nα, Nα, Nα‐ trimethyl‐L‐histidine, “selenoneine” in tuna blood had a strong antioxidant capacity and bound to hemoglobin and myoglobin (Mb), to protect them from iron autoxidation, and reacted with radicals and methylmercury (MeHg). Cell growth of human and zebrafish cultured cells were enhanced in the presence of selenoneine at 5–100 nM, and GPx1 gene expression was induced in dose‐dependent manner. Administration of selenoneine to yellowtail fish reduced ROS and met‐Mb formation, and the meat color change in dark muscle was protected. Selenoneine was incorporated into human embryonic kidney HEK293 transiently overexpressed with organic cations/carnitine transporter‐1 (OCTN1) and zebrafish embryo cells by OCTN1. The Km value of selenoneine uptake was determined to be 13.0 μM in OCTN1‐oeverexpressed HEK293 cells and 9.5 μM in zebrafish erythrocytes, respectively, indicating that selenoneine is the most specific substrate for the OCTN1 in the human and zebrafish cells. When such OCTN1‐expressing cells and embryos were exposed to MeHg–cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg was enhanced by the presence of selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1‐deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Therefore, the selenoneine‐mediated OCTN1 transport system accelerates secretory extracellular lysosomal vesicle formation and thus the demethylation of MeHg.

The novel selenium (Se)‐containing imidazole compound, 2‐selenyl‐trimethyl‐histidine, selenoneine, has been identified from the blood and tissues of the tuna (Yamashita et al, JBC, 285, 18134–18138, 2010). This compound had a strong radical scavenging activity, and had a protective function under oxidative stress conditions. In order to elucidate the molecular mechanism of DNA repair by Se, DNA damage response to γ‐irradiation was characterized in zebrafish embryos (8 hpf) in the presence of selenoneine at 10–100 nM in culture medium at 0.1–8 Gy. Although apoptosis was induced and ionizing radiation had no discernable effects on overall growth and morphology, TUNEL‐positive apoptotic cells were detected in brain, eye and spinal cord. Such apoptosis in CNS was reduced by the treatment of selenoneine at 25 nM after γ‐irradiation. CDC48/VCP/p97, which promotes both ubiquitin‐proteasome system and autophagy, was phosphorylated at Ser‐784 in response to DNA damage, and the treatment of selenoneine blocked CDC48 phosphorylation, suggesting that DNA double strand break by ionizing radiation might be reduced by selenoneine radical scavenging function. Knockdown of CDC48 by antisense morpholino oligos enhanced abnormal morphogenesis and apoptosis in CNS by γ‐irradiation. Therefore, Se redox mechanism by selenoneine and CDC48‐mediated autophagy had a protective function against ionizing radiation.

The selenium-containing imidazole compound selenoneine (2 selenyl N-alpha, N-alpha, N-alpha-trimethyl L histidine) was injected into yellowtail intravenously, and a fillet was prepared 18 h after the injection. The selenoneine concentration in the red blood cells (RBCs) and red muscle were higher in the selenoneine-injected fish than in the control (water-injected) fish. A close correlation was detected between the selenoneine concentration in the RBCs and that in red muscle. In addition, a negative correlation was noted between the selenoneine concentration in the red muscle and the ROS level in the plasma. After the fish were killed, the fillets were stored on ice for 72 h, and meat slices were then prepared and stored for an additional 24 h at 4 degrees C. The change in meat color in the selenoneine-injected fish was delayed, and the a* value of the red muscle was conserved compared with that in the control fish. Therefore, selenoneine was found to be responsible for the prevention of met-Mb formation in the red muscle of yellowtail.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.