Determination of Arsenic Species in Edible Insects by LC-ICP-MS.

BACKGROUND: Edible insects may contain arsenic. Analysis of arsenic species is necessary in order to accurately assess arsenic exposure. OBJECTIVE: An analytical method was validated and used to determine and quantitate arsenic species in edible insects. METHODS: Arsenic species were extracted from edible insects by heating at 100°C in 0.3 mol/L nitric acid. The concentration of arsenic species was then determined by LC-inductively coupled plasma-mass spectrometry (LC-ICP-MS) using an octadecylsilane (ODS) column with a mobile phase containing an ion-pair reagent. RESULTS: The LOD (0.007-0.012 mg/kg), LOQ (0.021-0.038 mg/kg), repeatability (1.2-3.2%), intermediate precision (2.8-4.5%), and trueness (recoveries 97-102% based on spiked samples) of the proposed method were satisfactory for inorganic arsenic, dimethylarsinic acid (DMA), and arsenobetaine (AB) in edible insects. Total arsenic was detected in all samples obtained in Japan (Asian forest scorpion, diving beetles, giant water bug, grasshoppers, June beetles, mole crickets, male rhino beetle, female rhino beetle, sago worms, and silkworm pupae) and consisted of mostly inorganic arsenic. Beetles in particular showed relatively high levels. CONCLUSION: Arsenic content varies among edible insect species. Feed control is important, as arsenic concentrations in edible insects may be feed dependent. HIGHLIGHTS: Arsenic species in edible insects were analyzed by LC-ICP-MS using an ODS column with a mobile phase containing an ion-pair reagent. Inorganic arsenic was detected in most samples, with concentrations ranging from <0.04 to 29.3 mg/kg.

Identification of Monomethylmonothioarsonic Acid as the Major Thioarsenical Generated During Extraction Processes for Arsenic Species Analysis.

Acid extraction is commonly used to analyze arsenic species in rice. During the extraction process, spiked monomethylarsonic acid (MMA) is often transformed into different compounds. A similar phenomenon is observed in the arsenic speciation analysis of seafood. To identify these compounds, we analyzed a previously prepared extract using liquid chromatography-time-of-flight/mass spectrometry in differential analysis and liquid chromatography-inductively coupled plasma-MS. The compound was identified as monomethylmonothioarsonic acid (MMMTA), a thioarsenical, which is estimated to be more cytotoxic than MMA. As MMMTA was readily produced by bubbling hydrogen sulfide through MMA, this suggests that MMA reacts with sulfur in rice during the extraction process. Our data also suggested that dimethylarsinic acid could be transformed into another compound, although the generation rate was low. For reliable arsenic speciation analyses, the transformation of arsenic compounds during extraction must be avoided. This study demonstrates that arsenic compounds can be transformed by dilute acid extraction.

Mutations equivalent to Drosophila mago nashi mutants imply reduction of Magoh protein incorporation into exon junction complex.

Pre-mRNA splicing imprints mRNAs by depositing multi-protein complexes, termed exon junction complexes (EJCs). The EJC core consists of four proteins, eIF4AIII, MLN51, Y14 and Magoh. Magoh is a human homolog of Drosophila mago nashi protein, which is involved in oskar mRNA localization in Drosophila oocytes. Here we determined the effects of Magoh mutations equivalent to those of Drosophila mago nashi mutant proteins that cause mis-localization of oskar mRNA. We found that Magoh I90T mutation caused mis-localization of Magoh protein in the cytoplasm by reducing its binding activity to Y14. On the other hand, G18R mutation did not affect its binding to Y14, but this mutation reduced its association with spliced mRNAs. Our results strongly suggest that Magoh mutations equivalent to Drosophila mago nashi mutants cause improper EJC formation by reducing incorporation of Magoh into EJC.

Extending the Limbus-to-Cannula Distance to 6.0 mm During Pars Plana Vitrectomy in Highly Myopic Eyes.

PURPOSE: To evaluate the utility of extending the limbus-to-cannula distance to 6.0 mm during pars plana vitrectomy for highly myopic eyes. METHODS: Four eyes with axial lengths exceeding 31.0 mm, that underwent 25-gauge pars plana vitrectomy were retrospectively evaluated. Assuming that cannulas were inserted 3.5 mm and 6.0 mm from the corneal limbus, the distance from the cannula to the fovea (CF distance) was preoperatively evaluated using anterior segmental optical coherence tomography. Surgical complications were also investigated. RESULTS: The CF distance was shortened by 1.22 ± 0.05 mm and 1.22 ± 0.09 mm on the temporal and nasal sides, respectively, by inserting the cannula at 3.5 mm to 6.0 mm from the corneal limbus. As per the preoperatively measured CF distance, one of the cannulas was inserted 6.0 mm from the corneal limbus in three eyes. Their cannulas were confirmed to be inserted at the pars plana, and no surgical complications associated with this technique were observed. CONCLUSION: Extending the limbus-to-cannula distance to 6.0 mm during pars plana vitrectomy could be one of the options to reach the posterior pole in highly myopic eyes. A preoperatively measured CF distance can be a clinical criterion in determining the cannula position.

Mechanistic Insights of Aberrant Splicing with Splicing Factor Mutations Found in Myelodysplastic Syndromes.

Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3' splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.

Association between Bathing and Survival in Patients with Advanced Cancer in Their Last Days of Life: A Prospective Cohort Study.

Background: Although many Japanese patients wish to take a bath in their last days, the safety of bathing for patients with a prognosis of a few days is not known. Objective: To examine whether taking a bath affects the survival of advanced cancer patients with prognoses of a few days. Design: A single-center prospective cohort study. Setting/Subject: Advanced cancer patients in their last days of life in a palliative care unit of a Japanese hospital. We compared patients who took baths with those who did not. The primary endpoint was 24-hour survival rate. Result: Among 110 patients eligible for this prospective study, 89 (72%) met the inclusion criteria. Forty-eight patients (43%, 223 person-days) were eligible for analysis. A total of 28 patient-days were classified into the bathing group, and 192 patient-days were classified into the nonbathing group. After propensity score matching, the 24-hour death rate was 10.7% in the bathing group and 8.0% in the nonbathing group, respectively (mean difference 2.8% with 95% confidence interval of -11.2% to 16.8%, p = 0.65). Conclusion: Taking a bath does not appear to bear a significant association with shortening of life among advanced cancer patients in their last days of life.

AMP-activated protein kinase regulates ß-catenin protein synthesis by phosphorylating serine/arginine-rich splicing factor 9.

ß-catenin is a multi-functional protein with a central role in regulating embryonic development and tissue homeostasis. The abnormal accumulation of ß-catenin, due to disrupted ß-catenin degradation or unregulated ß-catenin synthesis, causes the development of cancer. A recent study showed that the overexpression of proto-oncogene serine/arginine-rich splicing factor 9 (SRSF9) promotes ß-catenin accumulation via binding ß-catenin mRNA and enhancing its translation in a manner that is dependent on the mechanistic target of rapamycin (mTOR). However, the regulation of the interaction between SRSF9 and mRNA of ß-catenin remains unclear. Here, we show that AMP-activated protein kinase (AMPK) phosphorylates SRSF9 at the RNA-interacting SWQDLKD motif that plays a major role in determining substrate specificity. The phosphorylation by AMPK inhibits the binding of SRSF9 to ß-catenin mRNA and suppresses ß-catenin protein synthesis caused by SRSF9 overexpression without changing the ß-catenin mRNA levels. Our findings suggest that AMPK activators are potential therapeutic targets for SRSF9-derived overproduction of ß-catenin in cancer cells.

Determination of Inorganic Arsenic in Fish Oil and Fish Oil Capsules by LC-ICP-MS.

BACKGROUND: As inorganic arsenic is a highly toxic compound, its concentration in foods should be determined. OBJECTIVE: Develop an analytical method for determining inorganic arsenic in fish oil and fish oil capsules. METHOD: Inorganic arsenic was extracted from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The concentration of inorganic arsenic in fish oil was determined by liquid chromatography (LC) inductively coupled plasma (ICP) MS using an octadecylsilane (ODS) column with a mobile phase containing an ion-pair reagent. RESULTS: The LOD (0.005, 0.004 mg/kg), LOQ (0.016, 0.011 mg/kg), repeatability (4.2, 3.5%), intermediate precision (5.4, 3.5%), and trueness (recoveries 94-109% based on spiked samples) of the proposed method were satisfactory for inorganic arsenic in fish oil and fish oil capsules. A low level of inorganic arsenic was detected only in anchovy oil among all fish oil samples that were used in this study. Inorganic arsenic levels were below the quantitation limit in all fish oil capsules. CONCLUSIONS: Inorganic arsenic was determined after extraction from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The level of inorganic arsenic in all fish oil samples examined in this study was lower than 0.1 mg/kg of the maximum level defined in the Codex. HIGHLIGHTS: Arsenic speciation in fish oil and fish oil capsules was analyzed by LC-ICP-MS using an ODS column with a mobile phase containing an ion-pair reagent. A low level of inorganic arsenic was detected only in anchovy oil. No inorganic arsenic was detected in fish oil capsules.

Whole-Exome Sequencing-Based Approach for Germline Mutations in Patients with Inborn Errors of Immunity.

PURPOSE: Owing to recent technological advancements, using next-generation sequencing (NGS) and the accumulation of clinical experiences worldwide, more than 420 genes associated with inborn errors of immunity (IEI) have been identified, which exhibit large genotypic and phenotypic variations. Consequently, NGS-based comprehensive genetic analysis, including whole-exome sequencing (WES), have become more valuable in the clinical setting and have contributed to earlier diagnosis, improved treatment, and prognosis. However, these approaches have the following disadvantages that need to be considered: a relatively low diagnostic rate, high cost, difficulties in the interpretation of each variant, and the risk of incidental findings. Thus, the objective of this study is to review our WES results of a large number of patients with IEI and to elucidate patient characteristics, which are related to the positive WES result. METHODS: We performed WES for 136 IEI patients with negative conventional screening results for candidate genes and classified these variants depending on validity of their pathogenicity. RESULTS: We identified disease-causing pathogenic mutations in 36 (26.5%) of the patients which were found in known IEI-causing genes. Although the overall diagnostic rate was not high and was not apparently correlated with the clinical subcategories and severity, we revealed that earlier onset with longer duration of diseases were associated with positive WES results, especially in pediatric cases. CONCLUSIONS: Most of the disease-causing germline mutations were located in the known IEI genes which could be predicted using patients' clinical characteristics. These results may be useful when considering appropriate genetic approaches in the clinical setting.

AMP-activated protein kinase regulates alternative pre-mRNA splicing by phosphorylation of SRSF1.

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic processes and activating catabolic processes. Recent studies have demonstrated that metformin, which is an AMPK activator, modifies alternative precursor mRNA (pre-mRNA) splicing. However, no direct substrate of AMPK for alternative pre-mRNA splicing has been reported. In the present study, we identified the splicing factor serine/arginine-rich splicing factor 1 (SRSF1) as a novel AMPK substrate. AMPK directly phosphorylated SRSF1 at Ser133 in an RNA recognition motif. Ser133 phosphorylation suppressed the interaction between SRSF1 and specific RNA sequences without altering the subcellular localization of SRSF1. Moreover, AMPK regulated the SRSF1-mediated alternative pre-mRNA splicing of Ron, which is a macrophage-stimulating protein receptor, by suppressing its interaction with exon 12 of Ron pre-mRNA. The findings of this study revealed that the AMPK-dependent phosphorylation of SRSF1 at Ser133 inhibited the ability of SRSF1 to bind RNA and regulated alternative pre-mRNA splicing.

Intratumoral CD8+ Lymphocyte Infiltration as a Prognostic Factor and Its Relationship With Cyclooxygenase 2 Expression and Microsatellite Instability in Endometrial Cancer.

OBJECTIVE: Microsatellite instability (MSI) is caused by a defective DNA mismatch repair system. Colorectal cancer in MSI-positive patients is characterized by an increased number of tumor-infiltrating lymphocytes. On the other hand, it has recently been reported that cyclooxygenase 2 (COX-2) suppresses antitumor immunity. The objectives of the present study were to clarify the relationships among MSI status, COX-2 expression, and antitumor immune status and to verify impact of these factors on the prognosis of endometrial cancer. METHODS: The data of 123 patients with endometrial cancer were analyzed. The numbers of tumor-infiltrating CD8 T lymphocytes within cancer cell nests (TILs), as a representative of the antitumor immunity, and COX-2 expression levels in the tumor cells were analyzed by immunohistochemical staining. Microsatellite instability was evaluated by polymerase chain reaction analysis for 11 markers. Fisher exact probability test, Kaplan-Meier method, and proportional hazards analysis were used for the statistical analyses. RESULTS: The MSI-positive tumors showed significantly higher grades (G2 or G3) and significantly larger numbers of TILs than did the MSI-negative tumors. The COX-2-high group showed significantly fewer TILs than did the COX-2-low group. Multivariate analysis identified a low number of TILs (<10), positive lymph node involvement, and high tumor malignancy grade as factors independently associated with poor prognosis. The prognosis was significantly poorer in the patients with MSI-positive tumors with high COX-2 expression than in those with MSI-positive tumors showing low COX-2 expression. CONCLUSIONS: The number of TILs, which was increased by MSI and decreased by COX-2 expression, was associated with a poorer prognosis in patients with endometrial cancer. We also propose that COX-2 may block MSI-activated TILs in the tumor microenvironment.

Reversed Phase Column HPLC-ICP-MS Conditions for Arsenic Speciation Analysis of Rice Flour.

New measurement conditions for arsenic speciation analysis of rice flour were developed using HPLC-ICP-MS equipped with a reversed phase ODS column. Eight arsenic species, namely, arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), dimethylarsinic acid (DMAA), trimethylarsine oxide (TMAO), tetramethylarsonium (TeMA), arsenobetaine (AsB) and arsenocholine (AsC), were separated and determined under the proposed conditions. In particular, As(III) and MMAA and DMAA and AsB were completely separated using a newly proposed eluent containing ammonium dihydrogen phosphate. Importantly, the sensitivity changes, in particular those of As(V) and As(III) caused by coexisting elements and by complex matrix composition, which had been problematical in previously reported methods, were eliminated. The new eluent can be applied to C8, C18 and C30 ODS columns with the same effectiveness and with excellent repeatability. The proposed analytical method was successfully applied to extracts of rice flour certified reference materials.

Effects of polishing, cooking, and storing on total arsenic and arsenic species concentrations in rice cultivated in Japan.

The effects of polishing, cooking, and storing on total arsenic (As) and As species concentrations in rice were studied adopting typical Japanese conditions. Total and inorganic As levels in three white rice samples polished by removing 10% of bran by weight were reduced to 61-66% and 51-70% of those in brown rice. The As levels in the white rice after three washings with deionized water were reduced to 81-84% and 71-83% of those in raw rice. Rinse-free rice, which requires no washing before cooking because bran remaining on the surface of the rice was removed previously, yielded an effect similar to that of reducing As in rice by washing. Low-volume cooking (water:rice 1.4-2.0:1) rice to dryness did not remove As. The As content of brown rice stored in grain form for one year was stable.

Speciation and determination of inorganic arsenic in rice using liquid chromatography-inductively coupled plasma/ mass spectrometry: collaborative study.

An analytical method to speciate two inorganic As forms [arsenite, As(lll) and arsenate, As(V)] in indica and japonica types of rice (both husked and polished) and determine the inorganic As concentration as the sum of these two was internationally validated. The method can additionally determine two organic As compounds, monomethylarsonic acid and dimethylarsinic acid, in rice as separate LC peaks. The method is based on LC separation and inductively coupled plasma (ICP)-MS detection. The method was evaluated through the International Union of Pure and Applied Chemistry/lnternational Organization for Standardization/AOAC harmonized protocol. Sixteen laboratories from four countries participated in the study, and 13 laboratories returned valid data. Twenty test portions of 10 blind duplicates of indica and japonica type rice samples (both husked and polished) were used in this study. Repeatability RSD (RSDr) and reproducibility RSD (RSDR) were calculated at five concentrations of total inorganic As between 0.03 and 0.68 mg/kg. The RSDr was in a range of 3.8 to 7.7% and the RSDR was in a range of 10 to 36%. These performance characteristics were found to be sufficient for determination of inorganic As at or higher than 0.03 mg/kg. Applicability of the method was estimated to be in a range of 0.02-2.0 mg/kg.

Determination of sixteen elements and arsenic species in brown, polished and milled rice.

The concentrations of 16 elements in 10 rice flour samples and the distribution of the elements in the rice grains from which the flour were made were determined by ICP-MS and ICP-OES after microwave-assisted digestion of the samples. Arsenic speciation analysis was carried out by HPLC-ICP-MS following heat-assisted extraction of the sample. The concentrations of inorganic As (As(III) and As(V)), monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) and their distribution in the rice grains were determined. Portions of the brown rice were polished/milled to different degrees to yield milled off samples and polished rice samples. All samples were powdered and analyzed for 16 elements and for As species. The recoveries and mass balances for all elements in all samples showed good agreements with the starting materials. As(III), As(V), MMAA and DMAA were detected, and the sums of the concentrations of all species in the extract were 86-105% of the total As concentration in each case.

Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer.

The fibroblast growth factor receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was >or=11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of 15 non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC.

Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability-positive endometrial cancer.

PURPOSE: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). EXPERIMENTAL DESIGN: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tum or lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. RESULTS: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). CONCLUSION: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.