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By using the LC-MS/MS method developed by us, we determined the residual amounts of acaricides in honey samples commercially available in Tokyo from April 2015 to March 2021. The results of analyzing 127 honey samples, amitraz was detected in 85 samples at the level of 1.1-34.1 μg/kg. Propargite was detected in 3 samples at 2.4-3.8 μg/kg. None of them was beyond the Japanese MRLs or uniform limits. In these survey for 6 years, amitraz was detected in high rate throughout the year. But, the present results imply that amitraz has been used properly in actual bee-keeping because of no violation of MRL and less fluctuation in the detected levels. On the other hand, propargite was detected at the levels over LOQ in domestic honey samples for the first time in 2020, which may suggest a new trend of acaricide use in apiculture in Japan.

Pulmonary pleomorphic carcinoma is often refractory to chemotherapy and follows an aggressive clinical course. Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced lung cancer, and a few cases with pleomorphic carcinoma have been reported to show tumor shrinkage after therapy with ICIs. When treating patients with ICIs, patient selection is essential, and monitoring and management of immune‐related adverse events, including pneumonitis, are needed. We herein report a case of pulmonary pleomorphic carcinoma with preexisting interstitial pneumonia treated with pembrolizumab, antiprogrammed cell death 1 antibody. Our report highlights important considerations necessary when treating advanced pleomorphic carcinoma patients complicated with interstitial pneumonia. We also review the literature regarding the use of ICIs in such patients.

Immune checkpoint inhibitor (ICI) programmed death (PD)‐1/PD‐ligand 1 (PD‐L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti‐PD‐L1/PD‐1 Ab in tumor angiogenesis. In syngeneic mouse models, anti‐PD‐L1 Ab inhibited tumor angiogenesis and induces net‐like hypoxia only in ICI‐sensitive cell lines. In tumor tissue and serum of ICI‐sensitive cell line‐bearing mice, interferon‐γ (IFN‐γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD‐L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN‐γ stimulation in tumor cell lines correlated with the sensitivity of PD‐L1 blockade. The CXCL10/11 receptor CXCR3‐neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD‐L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti‐PD‐1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD‐1/PD‐L1 blockade and identify tumor‐derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity., Our results show the novel antiangiogenic function of PD‐1/PD‐L1 blockade and identify tumor‐derived angiostatic chemokines CXCL10/11 as a potent circulating biomarker of therapeutic sensitivity. These findings contribute to the further understanding of antitumor mechanisms mediated by immune checkpoint inhibitors and provide a major advance in personalized immunotherapy.

An analytical method based on LC-MS/MS was developed for the determination of asulam in livestock products. Asulam in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and octadecyl silylated silica gel (C18) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of Asulam from four types of livestock products (bovine muscle, bovine fat, bovine liver and milk) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 92.7-98.7%, with a relative standard deviation of 3.1-11.6%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

Residual antibacterial agents in 5909 animal and fishery products in Tokyo, Japan, were investigated over 17 consecutive years (2003-2019). Monitoring of 32 antibacterial agents (lincosamides, macrolides, penicillins, quinorones and tetracyclines) per product was accomplished via two steps: screening (by microbiological methods) and confirmation (by instrumental methods). Microbiological screening methods identified presumptive groups and determined semi-quantitative values. The instrumental methods quantified 81 residues of 11 different antibacterial agents in 72 samples. The screening strategy based on microbiological methods demonstrated the following: (i) the majority of the samples (over 99%) met Japanese regulations, (ii) using multiple methods provided a reliable inspection system with accurate quantitative values and (iii) there was a constant presence of tetracyclines and unexpected residues (lincomycin and norfloxacin) in various products. Thus, this long-term monitoring and screening strategy provided evidence that the frequencies and trends of residual antibacterial agents not only enhance food safety but also help to prevent antimicrobial resistance.

An analytical method has been developed and validated for determining 107 pesticide residues in dried red pepper using LC-MS/MS. LC method, the clean-up and sample dilution processes were examined to determine their impact on reducing the matrix effects. Clean up was performed using an ENVI-CarbIITM/PSA (300/600 mg, 6 mL) SPE cartridge. In the sample dilution process, eight-fold dilution was used. In the validation of the developed method at two concentrations (0.01 and 0.1 μg/g) for 107 pesticides, 96 pesticides showed recovery rates in the range of 70.1 to 112.6%, RSDs of repeatability of ≤11.5 and 3.4%, and RSDs of within-laboratory reproducibility of ≤24.3 and 19.9%. These values fulfill the criteria of the validation guidelines for pesticide residues in Japan. It is concluded that matrix effects and low recovery rates in the process of extraction are the main factors for values that do not conform to the criteria.

Background: Treatment of non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD) is limited because of the risk of its acute exacerbation (AE). Furthermore, the efficacy and safety of second-line chemotherapy for these patients is unclear.Patients and Methods: To investigate the efficacy and safety of second-line chemotherapy for NSCLC patients with ILD, we retrospectively reviewed the patients who were treated at our institute between April 2010 and December 2018.Results: Thirty-five patients received two or more regimens. Thirty-four patients were male and the median age at the initiation of second-line chemotherapy was 70 years. Almost all patients had a smoking history. Fourteen patients had adenocarcinoma and 15 had squamous cell carcinoma histology. Stages Ⅲ and Ⅳ were observed in 20 and 11 patients, respectively. With respect to the type of ILD, 12 patients had usual interstitial pneumonia (UIP). The overall response rate and disease control rate were 11.4% and 68.6%, respectively. The median progression-free survival and median overall survival were 4.1 months and 6.4 months, respectively. The AE of ILD was observed in eight patients, five of whom died. UIP and low % vital capacity were detected as significant risk factors for the AE of ILD.Conclusion: Second-line chemotherapy among patients with NSCLC complicated by ILD showed a certain effectiveness, but some patients experienced the AE of ILD, which may lead to death. The risk of the AE of ILD must be considered especially for patients with UIP and low %VC.

The last decade has witnessed several events that had a serious impact on people’s attitudes toward environmental sustainability regionally and globally. This chapter depicts how transboundary cooperative initiatives by states and nonstate actors tackle transboundary air pollution and climate change in East Asia. It also examines the opportunities and challenges we face in the very recent landscape shift toward carbon neutrality and the deepening concerns for climate emergencies. Multilateral cooperative institutions in East Asia have focused on the monitoring of air pollutants and information sharing of related policies and measures among member countries. It should also be noted that there are some transboundary coalitions of independent scholars and research-type NGOs in Northeast Asia who conduct joint research on the decarbonization of energy systems and disseminate up-to-date knowledge and information on decentralized nature-based renewable energy. For further development of transboundary cooperation in East Asia, opportunities exist as an increasing potential for multilateral policy dialogue beyond the borders and broadening partnerships for local and transboundary coalitions with global alliances on the one hand; and challenges in just and safe transition, decarbonization of overseas financing, and seeking energy resilience on the other.

This article provides empirical evidence of the emergence of new private governance forms through three case studies: transboundary air pollution, green supply chain, and energy transition in North...

In the field of food analysis and regulation, different instruments are used to determine the accuracy of quantification values. This is essential, as inconsistencies in values are commonly encountered. To visualize the degree of these discrepancies in each food matrix, we compiled a validation study based on a routine method developed in our laboratory, for 121 pesticides in six agricultural products, namely the grapefruit, potato, paprika, cabbage, spinach, and brown rice. These were analyzed by GC-MS/MS and LC-MS/MS, and the results were compared mainly on the basis of trueness. According to the results of the validation study when using GC-MS/MS, of the 121 pesticides tested in each product class, the number of analytes that satisfied the criteria of the Japanese validation guidelines was 97 in grapefruit, 111 in potato, 110 in paprika, 118 in cabbage, 111 in spinach, and 63 in brown rice. In contrast, in the analysis of the same samples by using LC-MS/MS, the number of analytes that satisfied the criteria of the validation guidelines was 50 in grapefruit, 114 in potato, 103 in paprika, 112 in cabbage, 100 in spinach, and 103 in brown rice. Inconsistences in the differences of trueness were mainly attributed to matrix effects of each instrument, as well as to food matrices, of which the most diverged matrix was that of brown rice (over 20%).

The determination of antibacterial agents for animals in swine muscles was improved by microbiological screening and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. In the first instance, the residual drugs were extracted from the samples using the Na2EDTA-McIlvaine buffer (pH 6.0). Subsequently, the agents were purified utilizing a PLS-3 cartridge and extracted with acetonitrile. Considering the microbiological methods, the sensitivities of the investigated drugs were higher and the test plate conditions were improved using a new reference organism Geobacillus stearothermophilus. As a result, a microbiological screening approach able to detect 33 drugs at MRL was developed in Japan. Remarkable, no false positives were detected. Moreover, the same preparation method enabled rapid and reliable microbiological screening, resulting in efficient screening with no undeterminable results.

Background: The combination of anti-PD-1/PD-L1 antibody with chemotherapy has been approved for the first-line therapy of lung cancer. However, the effects against malignant mesothelioma (MPM) and the immunological mechanisms by which chemotherapy enhances the effect of targeting PD-1/PD-L1 in MPM are poorly understood. Materials and Methods: We utilized syngeneic mouse models of MPM and lung cancer and assessed the therapeutic effects of anti-PD-1 antibody and its combination with cisplatin (CDDP) and pemetrexed (PEM). An immunological analysis of tumor-infiltrating cells was performed with immunohistochemistry. Results: We observed significant therapeutic effects of anti-PD-1 antibody against MPM. Although the effect was associated with CD8+ and CD4+ T cells in tumors, the number of Foxp3+ cells was not reduced but rather increased. Consequently, combination with CDDP/PEM significantly enhanced the antitumor effects of anti-PD-1 antibody by decreasing numbers of intratumoral myeloid-derived suppressor cells (MDSCs) and vessels probably through suppression of VEGF expression by CDDP+PEM. Conclusions: The combination of anti-PD-1 antibody with CDDP+PEM may be a promising therapy for MPM via inhibiting the accumulation of MDSCs and vessels in tumors.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity of thoracic sarcomas with an undifferentiated rhabdoid morphology and SMARCA4 inactivation. Regardless of some reports about the histopathological findings so far, there have been only a few reports about the cytological features. In this article, we present the pathological features of 2 SMARCA4-DTS cases, including the cytological findings. Histopathologically, the tumor cells showed atypical loosely cohesive large epithelioid cells focally with geographic necrosis. Some cells were characterized by rhabdoid cells. Both patients showed intrathoracic masses with a history of smoking, and loss of SMARCA4 expression was confirmed with histopathological specimens. Immunohistochemically, tumor cells of both cases were at least focally positive for cytokeratin, CD34, CD99, synaptophysin, SOX2, and SALL4. In addition, tumor cells demonstrated significantly reduced expression of BRG1/SMARCA4 and SMARCA2. In conclusion, SMARCA4-DTS should be taken into consideration in the differential diagnosis of tumors with undifferentiated rhabdoid morphology involving the thoracic region.

The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96–100% whereas the sensitivity ranged 56–67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41–46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83–95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50–63% in T790M, and in 63–100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR.

In this study, the staphylococcal enterotoxin type A (SEA) contaminant was quantified in cow milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin's presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 µg kg-1 conducted with two samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The relative standard deviations of repeatability were 13-14% and the relative standard deviations of within-laboratory reproducibility were 13-18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The quantification limit was estimated to be 10 µg kg-1.

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSD

Objectives The clinical benefit of chemotherapy and the appropriate regimen for non-small-cell lung cancer (NSCLC) patients with interstitial lung disease (ILD) remain unclear. To fulfill this unmet medical need, we conducted a phase II study to elucidate the efficacy of S-1 in combination with carboplatin (CBDCA) in NSCLC patients with ILD. Materials and methods A total of 33 advanced or recurrent NSCLC patients with ILD were prospectively enrolled in this multicenter, open-label, phase II study. Every 4 weeks, CBDCA at a dose of AUC 5 on day 1 and S-1 at a dose of 80 mg/m2 daily for 14 days were administered. The primary endpoint was the investigator-assessed objective response rate. Results The median age at initiating chemotherapy was 70. Sixteen patients (48.5%) had squamous cell carcinoma histology. With respect to the types of ILD, the usual interstitial pneumonia pattern was dominant (66.7%). The median number of cycles administered was 3, and the overall response rate and disease control rate were 33.3% and 78.8%, respectively. The median progression-free survival, the median survival time and the 1-year survival rate were 4.8 months, 12.8 months and 51.4%, respectively. Acute exacerbation of ILD caused by chemotherapy was noted in 2 patients (6.1%). Conclusion This is the first prospective study designed to evaluate the efficacy of a specific chemotherapeutic regimen as the primary endpoint in patients with advanced NSCLC with ILD. The combination of S-1 with CBDCA may be a treatment option for advanced NSCLC patients with ILD (The clinical trial registration number: UMIN000011046).

We measured the residual amounts of chlorantraniliprole in various vegetables and fruits. Sample solutions were prepared according to our routine procedure based on the QuEChERS method and analyzed by LC-MS/MS. Performance characteristics were evaluated for 8 kinds of food samples by means of recovery tests of 5 replicates at the concentration of 10 ng/g. Recoveries and RSDs (％) ranged from 50.2 to 93.4％ and from 2.1 to 9.7％, respectively. Application of this method to survey 207 vegetables and 163 fruits gave detection rates of 8.2 and 1.2％, respectively. In vegetables, detection rates were high in okra (4 out of 10 samples), paprika (4 out of 23 samples) and tomato (2 out of 6 samples), and leaf vegetables such as lettuce, mizuna, spinach and wrinkled greens also contained high concentrations of chlorantraniliprole. The highest residual concentration was 571 ng/g in mizuna. The samples containing chlorantraniliprole seemed to be mainly from Asian countries, including samples of domestic Japanese origin. However, none of them contained more than the MRL, which suggests that the use of chlorantraniliprole has been properly controlled.

A LC-MS/MS simultaneous analytical method for screening 191 pesticide residues in limes had been developed and validated. Pesticides were extracted with acetonitrile from samples. Then mixed salts, which were anhydrous magnesium sulfate for dehydration, sodium carbonate for adjusting pH, and sodium chloride for salting out, were added to the sample. After centrifugation, supernatant was transferred to a tube. The sample solution was cleaned up using solid phase extraction (SPE) with C18/GC/PSA for the determination by LC-MS/MS. The developed method was improved the recovery rate of thiabendazole, which had a low recovery rate by the conventional method. Validation study, which was following the guidelines of the Ministry of Health, Labor and Welfare, were carried out at 0.01 and 0.1 μg g-1 to evaluate the method. The results of 175 pesticides out of 191 were with satisfactory. A total of 19 imported lime samples sold in Tokyo was analyzed to evaluate the method, then 18 samples contained pesticide residues below MRLs. The developed method is applicable for detection of pesticide residues in lime.

Highlight • Bone is one of major metastatic site in patients with genitourinary (GU) cancer. • Accurately predicting survival of patients with bone metastasis (BM) is essential. • This external validation study proved high predictive accuracy of B-FOM score. • B-FOM score is a simple scoring model based on five prognostic factors. • B-FOM score is higher accurate tool comparing to other previously reported scores., Objective We previously developed genitourinary (GU) cancer-specific scoring system for prediction of survival in patients with bone metastasis (the Bone-Fujimoto-Owari-Miyake [B-FOM] scoring model) based on five prognostic factors: the type of primary tumor (prostate cancer (PCa) vs renal cell carcinoma (RCC) and PCa vs urothelial carcinoma (UC)), poor performance status (PS), visceral metastasis, high Glasgow-prognostic score (GPS), elevated neutrophil-to-lymphocyte ratio (NLR). The aim of this study was to externally validate and further improve the performance of the B-FOM score. Methods The external validation cohort comprised 309 patients with GU cancer with bone metastasis from multiple institutions. Clinical factors were analyzed using Kaplan-Meier method and COX regression hazard model. Performance of a modified B-FOM score was compared to that of other scoring models by the Kaplan-Meier method and the area under the curve (AUC) of receiver operating characteristic curves. Results The median follow-up period of development and validation cohort were 25 and 17 months, respectively. Kaplan-Meier curve demonstrated that the type of primary tumor (RCC and UC vs PCa), poor PS, presence of visceral metastasis, high GPS, elevated NLR were significantly associated with shorter cancer-specific survival. Risk groups were successfully stratified by the modified B-FOM score classification. Moreover, the AUC of the modified B-FOM scoring model for predicting mortality at 6, 12, and 24 months were 0.895, 0.856, and 0.815, respectively, which were the highest among evaluated models. Conclusions The B-FOM scoring model is a simple and accurate prediction tool. By using this scoring model at the time of the diagnosis of bone metastasis in patients with GU cancers, an individualized optimal treatment strategy can be selected.

Background: Recent clinical trials indicate the combination treatment with immune checkpoint inhibitor and antiangiogenic drug improves the prognosis of lung cancer patients. However, its mechanism has been still unclear. We previously reported tumor tissue treated with anti-vascular endothelial growth factor (VEGF) antibody recruits the fibrocyte-like cells (FCs). We focus on the FCs as the key mediators of tumor immune microenvironment. Aims and Objectives: The purpose of this study is to investigate the roles of FCs in the combination therapy with anti-VEGF and anti-programmed death-ligand 1 (PD-L1) antibodies by using the subcutaneous mouse model of mesothelioma. Method: Mouse mesothelioma cell line (AB1-HA) bearing mice were treated with anti-VEGF and anti-PD-L1 antibodies. The number of tumor infiltrated immune cells was determined by immunohistochemistry. We confirmed the expression of immune checkpoint molecules and costimulatory molecules in mouse lung FCs by flowcytometry. To evaluate the roles of FCs on the effect of anti-PD-L1 antibody, we implanted the mouse lung FCs near the tumor tissue. Results: The combination therapy with low dose anti-VEGF and PD-L1 antibody suppressed the tumor progression more than monotherapy. We confirmed low dose anti-VEGF antibody increases the tumor infiltrated FCs. Mouse FCs expressed PD-L1, and costimulatory molecules CD86. The FCs implantation improved the therapeutic effect of anti-PD-L1 antibody, and increased the tumor infiltrated CD8+ T cells. Conclusions: The combination blockage of VEGF and PD-L1 has potential to improve the anti-tumor immunity via recruiting PD-L1+, CD86+ FCs and enhancing its antigen presentation.

Fibrocytes, a distinct population of collagen-producing, monocyte-derived cells, are involved in wound healing as well as fibrotic diseases. Recently, fibrocytes have been revealed to play a role in the tumor microenvironment, particularly under antiangiogenic therapy. In addition, combination cancer immunotherapy with immune checkpoint inhibitor and antiangiogenic agents have been developed for various cancers in the clinical setting, although the immunological background is not clear. In the current study, we aimed to determine the function of fibrocytes in tumor immunity induced by immune checkpoint inhibitor therapy. Human and murine fibrocytes were generated from PBMCs and lungs, respectively. The expression of costimulatory and inhibitory molecules on fibrocytes was examined by flow cytometry. The stimulation of CD8+ T cells by fibrocytes was examined in MLRs with a 3H-thymidine incorporation assay. Fibrocytes expressed CD80low and CD86high as a costimulatory molecule, and expressed PD-L1high, but not PD-L2, as a coinhibitory molecule. Without any stimulation, fibrocytes strongly enhanced the proliferation of CD8+ T cells in mice and humans. Treatment with anti-CD86 and -CD54 Abs inhibited the growth of CD8+ T cells induced by fibrocytes. Anti–PD-L1 Ab further enhanced the proliferation of CD8+ T cells, even in the OVA-specific MLR with OT-1Rag−/− mice. Importantly, fibrocytes derived from PBMCs of patients with lung adenocarcinoma or murine MC38 tumors augmented the proliferation of CD8+ T cells with PD-L1 blockade. These results suggest that fibrocytes infiltrating tumor sites may play a role in the antitumor immunity mediated by CD8+ T cells when the activity is further enhanced by PD-L1/PD-1 blockade.

In this study, we developed a reference labelled protein containing the partial amino acid sequence of botulinum neurotoxin type A (BoNTA). We also applied it as an internal standard to detect specific and non-toxic peptides originated from BoNTA in honey with the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Original proteins in the honey sample were collected through a two-step process that included solubilisation and trichloroacetic acid (TCA) precipitation. Solubilisation by adding water enabled processing of proteins in honey. TCA precipitation collected proteins without specific binding. The combination of protein alkylation and an appropriate enzyme-to-protein ratio ensured feasibility of tryptic digestion. A desalting process eliminated a large amount of salts and other tryptic peptides in the honey sample. The use of the reference labelled protein enabled compensation for tryptic digestion efficiency and electrospray ionisation efficiency based on LC-MS/MS measurement. After the peptide selection and protein BlastP analysis, five unique peptides were chosen. The non-toxic peptides originating from BoNTA were reliably detected using LC-MS/MS based on a multiple-reaction monitoring mode. Detection of several peptides ensured screening of BoNTA in honey samples. Based on the responses, the proteotypic peptide LYGIAINPNR was selected as the quantitative peptide. Due to maintaining the relative ion ratios, the selective transition completely identified the non-toxic peptides. The intensity of the transitions established a detection limit of BoNTA estimated to be 9.4 ng mL

(Objectives) Transurethral Ureterolithotripsy (TUL) has become an increasingly more common treatment for ureteric stones since the reduction in ureteroscope diameter and other device improvements. At the same time, TUL sometimes shows postoperative febrile urinary tract infection (fUTI) with severe complications. Therefore we investigated the occurrence and risk factors of fUTI in our hospital, and assessed the effect of antibiotic prophylaxis prior to TUL. (Materials and methods) The subjects were 260 patients who underwent TUL in our department during the period from January 2011 to October 2014. We retrospectively reviewed the data of those who developed postoperative fUTI and identified the risk factors of postoperative fUTI. From November 2014 to August 2016, we enrolled 110 patients undergoing TUL with one or more risk factors in a prospective clinical trial of prophylactic oral levofloxacin (500 mg) for 1 week before TUL. The chi-squared test, Mann-Whitney U-test, and logistic regression analysis were used for data analysis (significance level of 0.05). (Results) Postoperative fUTI occurred in 43 (16.5%) of 260 patients. The risk factors of postoperative fUTI included preoperative pyelonephritis (P=0.02), preoperative ureteral stent placement (P=0.017), and operative time90 min (P=0.005). Operative time was correlated with and could be substituted for pre-TUL stone size (P0.0001). Chemopreventive therapy before TUL in patients with preoperative pyelonephritis, preoperative ureteral stent placement, or stones of20 mm significantly reduced their risk of fUTI (P=0.012). (Conclusions) The use of antibiotic prophylaxis significantly reduces the risk of postoperative fUTI in patients with preoperative pyelonephritis, preoperative ureteral stent placement, or stones of20 mm.

The low-density lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific receptor tyrosine kinase (MuSK) form a tetrameric protein complex on the postsynaptic membrane at the neuromuscular junction (NMJ). Binding of agrin to LRP4 triggers phosphorylation of MuSK. Activated MuSK drives clustering of acetylcholine receptor (AChR). Wnt ligands also directly bind to MuSK to induce AChR clustering. MuSK anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. In addition, an extracellular proteoglycan, biglycan, binds to MuSK. Anti-MuSK autoantibodies (MuSK-IgG) are observed in 5–15% of autoimmune myasthenia gravis (MG) patients. MuSK-IgG blocks both ColQ-MuSK and LRP4-MuSK interactions. MuSK-IgG, LRP4, ColQ, and biglycan bind to the immunoglobulin-like domains 1 and 4 of MuSK. Lack of the effects of cholinesterase inhibitors in MuSK-MG patients is likely due to hindrance of ColQ-MuSK interaction by MuSK-IgG and subsequent deficiency of AChE observed in model mice, which, however, has not been proven in MuSK-MG patients. As ColQ enhances expression of membrane-bound MuSK, inhibition of ColQ-MuSK interaction by MuSK-IgG may account for lack of AChR clusters in MuSK-MG. We thus made passive transfer models using Colq+/+ and Colq−/− mice to dissect the effect of ColQ on AChR clustering in MuSK-MG. We found that MuSK-IgG-mediated suppression of LRP4-MuSK interaction, not of ColQ-MuSK interaction, caused defective AChR clustering. We also unexpectedly observed that both MuSK-IgG and ColQ suppressed agrin/LRP4/MuSK signaling in dose-dependent manners. Quantitative comparison revealed that MuSK-IgG blocked agrin-LRP4-MuSK signaling more than ColQ. We propose that attenuation of AChR clustering in MuSK-MG is due to hindrance of LRP4-MuSK interaction in the presence of agrin by MuSK-IgG.

Vitamin B12 deficiency leads to various symptoms such as neuropathy, growth retardation, and infertility. Vitamin B12 functions as a coenzyme for two enzymes involved in amino acid metabolisms. However, there is limited information available on whether amino acid disorders caused by vitamin B12 deficiency induce such symptoms. First, free amino acid levels were determined in vitamin B12-deficient Caenorhabditis elegans to clarify the mechanisms underlying the symptoms caused by vitamin B12 deficiency. Various amino acids (valine, leucine, isoleucine, methionine, and cystathionine, among others) metabolized by vitamin B12-dependent enzymes were found to be significantly changed during conditions of B12 deficiency, which indirectly affected certain amino acids metabolized by vitamin B12-independent enzymes. For example, ornithine was significantly increased during vitamin B12 deficiency, which also significantly increased arginase activity. The accumulation of ornithine during vitamin B12 deficiency constitutes the first report. In addition, the biosynthesis of spermidine from ornithine was significantly decreased during vitamin B12 deficiency, likely due to the reduction of S-adenosylmethionine as a substrate for S-adenosylmethionine decarboxylase, which catalyzes the formation of spermidine. Moreover, vitamin B12 deficiency also demonstrated a significant reduction in worm lifespan, which was partially recovered by the addition of spermidine. Collectively, our findings suggest that decreased spermidine is one factor responsible for reduced lifespan in vitamin B12-deficient worms.

We developed an analytical method for determining 15 antifungal drugs, 2 antiparasitic drugs, and 3 veterinary drugs in fish and livestock products using LC-MS/MS. First, 50% ethanol was added to their products, and the mixture was homogenized to reduce drug degradation. Thereafter, 20 drugs were extracted from the pretreated sample mixture using acetonitrile. Cleanup was performed using an alumina-N SPE cartridge. Finally, chromatographic separation was performed using a fully porous octadecyl silanized silica column. The new method is applicable to fish in which the matrix hampers accurate analysis. It was validated on 8 fish and livestock products. Drug recovery rates ranged from 70.2 to 109.3%, RSDs of repeatability were

Although first-line chemotherapy is highly sensitive against small-cell lung cancer (SCLC), most patients subsequently experience disease progression. Topotecan is the standard therapy for sensitive-relapsed SCLC patients, and subgroup analysis of a randomized phase III trial suggests that amrubicin is effective for refractory-relapsed SCLC. However, because of the lack of the evidence based on clinical trials, the effectiveness of systemic chemotherapy for relapsed SCLC patients with idiopathic interstitial pneumonias (IIPs) is unclear. In the presentstudy, 17 relapsed SCLC patients with IIPs who received a paclitaxel (PTX)-containing regimen as a second-line chemotherapy were retrospectively reviewed. The overall response rate and the disease control rate of the PTX-containing regimens were 29.4 and 47.1%, respectively. The median progression-free survival and the overall survival of the regimens were 2.7 months [95% confidence interval (CI), 1.6-3.6 months] and 3.6 months (95% CI, 2.3-14.0 months), respectively. Grade 3-4 neutropenia and febrile neutropenia occurred in 12 (70.6%) and 2 (11.8%) patients, respectively. During the treatment period, acute exacerbation (AE) of IIPs was observed in five patients (29.4%). Treatment-associated fatality was observed in 1 patient with febrile neutropenia and in 1 patient with AE of IIPs. PTX had promising anti-tumor activity against refractory-relapsed SCLC with IIPs. However, the survival benefit of the treatment was limited because of the high incidence of AE of IIPs and treatment-related death.