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Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins.The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP.Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates.The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT2A and α2-adrenergic receptors on platelets and suppresses platelet aggregation mediated by ADP, increased by either 5-HT or adrenaline. Thus, a detailed monitoring of bleeding is recommended for patients taking MTZ.

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in Na+/K+-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT

Proteomics refers to the large-scale study of proteins, providing comprehensive and quantitative information on proteins in tissue, blood, and cell samples. In many studies, proteomics utilizes liquid chromatography-mass spectrometry. Proteomics has developed from a qualitative methodology of protein identification to a quantitative methodology for comparing protein expression, and it is currently classified into two distinct methodologies: quantitative and targeted proteomics. Quantitative proteomics comprehensively identifies proteins in samples, providing quantitative information on large-scale comparative profiles of protein expression. Targeted proteomics simultaneously quantifies only target proteins with high sensitivity and specificity. Therefore, in biomarker research, quantitative proteomics is used for the identification of biomarker candidates, and targeted proteomics is used for the validation of biomarkers. Understanding the specific characteristics of each method is important for conducting appropriate proteomics studies. In this review, we introduced the different characteristics and applications of quantitative and targeted proteomics, and then discussed the results of our recent proteomics studies that focused on the identification and validation of biomarkers of drug efficacy. These findings may enable us to predict the outcomes of cancer therapy and drug-drug interactions with antibiotics through changes in the intestinal microbiome.

IgA-producing lymphoplasmacytic lymphoma (LPL) is rare and IgH/c-myc translocation is rare in LPL. This is the first report of a case of IgA-producing LPL carrying t(8;14). An 86-year-old woman presented inguinal and intra-abdominal lymph node swelling, and lytic bone lesions in the lumbar vertebrae. A diagnosis of IgA-producing LPL was immunohistochemically made by inguinal lymph node biopsy. The serum IgA level was 1,180 mg/dL, which was revealed to be composed of IgA-λ monoclonal protein. Bone marrow chromosomal analysis demonstrated a complex abnormal karyotype, including t(8;14)(q24;q32), which was confirmed by FISH analysis. Abnormal lymphocytes positive for CD19, CD20, cyIgA, and cyλ were detected on flow cytometry analysis of marrow cells. Best supportive care was selected because of dementia and refractory urinary tract infection. Circulating lymphoplasmacytic cells with the same phenotype and karyotype were observed, and increased in number. The aggressive clinical course, including lytic bone lesions, may have been due to IgH/c-myc translocation or the nature of IgA-producing LPL.

Background: TNF inhibitors (TNFi) revolutionized the treatment of rheumatoid arthritis (RA), but have often developed anti-drug antibodies (ADrA) and also induced other autoimmune pathology1, 2). Objectives: To determine whether presence of anti-nuclear antibodies (ANA) predict the development of ADrA and clinical efficacy in RA patients treated with TNFi, infliximab (IFX) or adalimumab (ADA). Methods: A total of 92 RA patients, 38 cases treated with IFX and 54 cases with ADA, were prospectively enrolled. Observation period of IFX-treated group was 3 years, and that of ADA-treated group was 1 years. ANA were measured before and serially during the therapy. ANA were measured by indirect fluorescent assay using a Computer-aided microscope system with HEp20-10 cells. The concentrations of anti-IFX antibodies (HACA) and anti-ADA antibodies (AAA) were measured by radioimmunoassay at Sanquin Diagnostic Services, Netherlands. Clinical response to therapy was judged by EULAR response criteria based on Disease Activity Score (DAS) 28-CRP. Results: Thirteen (34.2%) of 38 patients in IFX group and 18 (33.3%) of 54 patients in ADA group became positive for HACA and AAA, respectively, during the treatment. All of the ADrA-positive patients were ANA-positive before the therapy. In contrast, none became ADrA-positive after treatment in 15 RA patients negative for ANA before therapy (6 cases in IFX group and 9 cases in ADA group), and the positive rate for ADrA was significantly different between ANA-positive and negative patients (p=0.0018). In addition, the positive rate for HACA was significantly higher in patients positive for ANA titers of ≥640 (p=0.034), but that for AAA was not related to ANA titers. Next, ANA became positive in 4 of 6 ANA-negative patients and ANA titers increased in 21 of 32 ANA-positive patients after IFX treatment. HACA positive rate was significantly higher in patients with ANA titers of ≥640 after IFX treatment (p=0.0045). ANA titers of patients in ADA group were mostly unchanging after treatment, and were not related to the appearance of AAA. Continuance rete of TNFi (IFX and ADA) treatment was significantly lower in ADrA-positive patients than in those negative (p=0.0066 and p=0.0127, respectively). In IFX group, patients with ANA titers of ≥160 before treatment and those with ANA titers of ≥ 320 after treatment positive showed worse EULAR treatment response (p=0.037 and p=0.033, respectively). In ADA group, 7 of 9 ANA-negative patients before treatment showed moderate or good EULAR response, but positive ANA both before and after treatment was not connected with to the clinical response. Conclusion: The presence of ANA before IFX or ADA is a risk factor for the appearance of ADrA, while ADrA did not appeared in any patient negative for ANA before treatment. ANA of high titers before and after IFX treatment predicted existence of ADrA and possibly leading to the treatment failure. References [1] Kalden JR, Shulze-Koops: Nat Rev Rheumatol, 2017, 13: 707-18. [2] Takase K, et al: Ann Rheum Dis, 2014, 73: 1695-99. Disclosure of Interests: Shunichi Kumagai Grant/research support from: Mitsubishi Tanabe Pharm, Eisai Co.Ltd., Ayano Mori: None declared, Toshiharu Saito: None declared, Miho Takahashi: None declared, Katsuhiko Yoneda: None declared, Yasuhiro Noda: None declared, Soushi Takahashi: None declared, Miwa Nishida: None declared, Saori Hatachi: None declared, Goh Tsuji: None declared

A diagnosis of acute monocytic leukemia (AML-M5) based on α-naphthyl butyrate esterase (α-NB) staining has some problems, because AML-M5 leukemic cells often show weak or faint positivity on α-NB staining. In these situations, some cases of AML-M5 tend to be misdiagnosed as AML-M0. Therefore, we evaluated the significance of weak or faint α-NB staining in AML-M5 diagnosed by flow cytometry (FCM). Nineteen AML cases in which leukemic cells were negative for naphthol AS-D chloroacetate esterase staining were studied. For FCM, we defined leukemic cells as having a monocytic nature when more than 10% of the leukemic cells were positive for at least one of the following antigens: CD4, CD11c, CD14, and CD64. The monocytic nature determined by FCM was consistent with positive or weak positivity on α-NB staining. Five of 6 cases in which leukemic cells exhibited faint positivity for α-NB staining could be diagnosed as AML-M5 by FCM, while negative α-NB staining was consistent with a diagnosis of AML-M0. These results suggest that AML-M5 should be taken into consideration even when leukemic cells are faintly positive for α-NB staining.

H3-scaled Acoustic Reduction Experiments (HARE) with a scale of 2.5 % was conducted for the prediction and reduction of lift-off acoustics for the H3 launch vehicle currently under development in Japan. The effect of vehicle elevation and shape of a movable launch platform was studied in the first testing campaign. Then, acoustic reduction based on acoustic shielding and water injection system was also studied in the second testing campaign. In this presentation, results of those testing campaign will be summarized. Then, in addition to the numerical study, acoustic design of the launch complex conducted in the H3 development will be presented.H3-scaled Acoustic Reduction Experiments (HARE) with a scale of 2.5 % was conducted for the prediction and reduction of lift-off acoustics for the H3 launch vehicle currently under development in Japan. The effect of vehicle elevation and shape of a movable launch platform was studied in the first testing campaign. Then, acoustic reduction based on acoustic shielding and water injection system was also studied in the second testing campaign. In this presentation, results of those testing campaign will be summarized. Then, in addition to the numerical study, acoustic design of the launch complex conducted in the H3 development will be presented.

The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17beta-estradiol (E(2)) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF)-stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E(2) or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E(2) and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E(2) and raloxifene may be mainly mediated by ERalpha.

A 38-year-old man was given a diagnosis of as sigmoid colon cancer and underwent sigmoid colectomy. Post-operative pathological staging was stage IIIb. He then underwent adjuvant chemotherapy. One year and 4 months after the surgery, CT scans revealed multiple liver and lung metastases. He was given mFOLFOX6+bevacizumab, which was changed later to FOLFIRI+bevacizumab. After these chemotherapies, he was admitted to the hospital due to sudden abdominal pain and high grade fever. Obstructive jaundice was initially diagnosed, but detailed study of initial CT revealed intragastric wall abscess. After the drainage of the abscess, his conditions improved. We speculated that the abscess formation was caused by mucosal damage due to bevacizumab.

Background Patients with adult T-cell leukemia (ATL) may have eyelid lymphoma, uveitis, or cytomegalovirus retinitis due to being immunocompromised. However, there have been few reports on the invasion of multiple ocular lesions. We treated 1 unusual ATL patient with uveitis in whom multiple ocular invasions were suspected. Case A woman in whom ATL was diagnosed 10 years previously complained of blurred vision and decreased visual acuity in the right eye. Anterior uveitis of the right eye was suspected. One week later the cells increased in the anterior chamber, and fibrin exudates and hyphema appeared. She was admitted to our hospital. Observations The visual acuity was 0.04 in the right eye and finger-counting from 30 cm in the left. She was treated with systemic steroid therapy. Inflammation disappeared, but both eyelids became swollen and multiple ocular lesions appeared. She was given carcinostatic therapy once more and the mass lesions decreased. Mass lesions appeared in the iris and in the bulbar conjunctiva. Computed tomography and magnetic resonance imagining (MRI) showed that the mass lesions extended to the right orbit and both nasal cavities. MRI also demonstrated choroidal thickening in the left eye. Conclusion This case documents that ATL cells may cause severe uveitis and invade multiple ocular tissues such as the iris, eyelid, choroids, and orbit.

Physical properties of liquid imidazole-borane complex were investigated to demonstrate their utility as aprotic polar solvents or liquid electrolytes appropriate for selective ion transport.