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Supplementary Figure 2 from An Essential Role of Alternative Splicing of c-myc Suppressor FUSE-Binding Protein–Interacting Repressor in Carcinogenesis

Elevated expression of c-myc has been detected in a broad range of human cancers, indicating a key role for this oncogene in tumor development. Recently, an interaction between FUSE-binding protein–interacting repressor (FIR) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and might be important for suppressing tumor formation. In this study, we showed that enforced expression of FIR induced apoptosis. Deletion of the NH2-terminal repression domain of FIR rescued the cells from apoptosis as did coexpression of c-Myc with FIR; thus, repression of Myc mediates FIR-driven apoptosis. Surprisingly, a splicing variant of FIR unable to repress c-myc or to drive apoptosis was frequently discovered in human primary colorectal cancers but not in the adjacent normal tissues. Coexpression of this splicing variant with repressor-competent FIR, either in HeLa cells or in the colon cancer cell line SW480, not only abrogated c-Myc suppression but also inhibited apoptosis. These results strongly suggest the expression of this splicing variant promotes tumor development by disabling FIR repression and sustaining high levels of c-Myc and opposing apoptosis in colorectal cancer. (Cancer Res 2006; 66(3): 1409-17)

Supplementary Figure 1 from An Essential Role of Alternative Splicing of c-myc Suppressor FUSE-Binding Protein–Interacting Repressor in Carcinogenesis

Background The interplay between cancer cells and stromal components, including soluble mediators released from cancer cells, contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we set out to identify key secreted proteins involved in PDAC progression. Methods We performed secretome analyses of culture media of mouse pancreatic intraepithelial neoplasia (PanIN) and PDAC cells using Stable Isotope Labeling by Amino acid in Cell culture (SILAC) with click chemistry and liquid chromatography-mass spectrometry (LC-MS/MS). The results obtained were verified in primary PDAC tissue samples and cell line models. Results Complement factor B (CFB) was identified as one of the robustly upregulated proteins, and found to exhibit elevated expression in PDAC cells compared to PanIN cells. Endogenous CFB knockdown by a specific siRNA dramatically decreased the proliferation of PDAC cells, PANC-1 and MIA PaCa-II. CFB knockdown induced increases in the number of senescence-associated-β-galactosidase (SA-β-gal) positive cells exhibiting p21 expression upregulation, which promotes cellular senescence with cyclinD1 accumulation. Furthermore, CFB knockdown facilitated downregulation of proliferating cell nuclear antigen and led to cell cycle arrest in the G1 phase in PDAC cells. Using immunohistochemistry, we found that high stromal CFB expression was associated with unfavorable clinical outcomes with hematogenous dissemination after surgery in human PDAC patients. Despite the presence of enriched CD8+ tumor infiltrating lymphocytes in the PDAC tumor microenvironments, patients with a high stromal CFB expression exhibited a significantly poorer prognosis compared to those with a low stromal CFB expression. Immunofluorescence staining revealed a correlation between stromal CFB expression in the tumor microenvironment and an enrichment of immunosuppressive regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We also found that high stromal CFB expression showed a positive correlation with high CD8+/Foxp3+ Tregs populations in PDAC tissues. Conclusions Our data indicate that CFB, a key secreted protein, promotes proliferation by preventing cellular senescence and is associated with immunological tumor promotion in PDAC. These findings suggest that CFB may be a potential target for the treatment of PDAC.

Background Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. Methods Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Results The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. Conclusions Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti‐far‐upstream element‐binding protein‐interacting repressor‐lacking exon2 (FIRΔexon2), anti‐sorting nexin 15, and anti‐spermatogenesis and oogenesis–specific basic helix–loop–helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large‐scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age‐matched HDs, the level of anti‐FIRΔexon2 Abs in GC patients was significantly higher (P, Anti‐FIRΔexon2 Ab is a predictive or overall survival marker for patients with gastric cancer.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score2.0, and 85 (100%) isolates were identified with a score1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke, transient ischemic attack , diabetes mellitus (DM), cardiovascular disease , chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than that of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.

Mass spectrometry (MS) is increasingly used in clinical medicine in Japan. In contrast to the successful application of MS in clinical microbiology, the adoption of MS-based assays for routine testing in clinical chemistry is very slow. In order to promote the significant benefits that MS platforms bring to laboratory medicine and patient care, the Japanese Society for Biomedical Mass Spectrometry (JSBMS) initiated a medical mass spectrometrist (MMS) certification program in 2013. As of Dec 2018, 308 persons from various medical specialties have been certified. We believe that they will play significant roles for expanding routine clinical MS usage in Japan.

Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.

Background Long-range PCR (LR-PCR) is used to enrich the target regions of the genome. This study aimed to establish the pipeline of targeted gene sequencing using LR-PCR and massively parallel sequencing (MPS). Methods The 14-kb-long MEFV gene, including the entire coding exons, was selected as a target gene and amplified using LR-PCR. The evaluated analytical factors were as follows: LR-PCR conditions, three types of post-PCR cleanup methods, and two types of MPS library preparation methods. Results With regard to LR-PCR conditions, Tks Gflex DNA polymerase at 7-min (30-s/kb) annealing/extension with 100-ng genomic DNA input had the highest yield. Regarding post-PCR purification methods, the magnetic beads-based method had high recovery and purity. In the MPS library preparation methods, the ligation-based method had a higher base coverage in the target (94.58%), uniformity of base coverage (99.95%), and target bases with no strand bias (97.40%). The exonic variants determined by Sanger sequencing were detected by both ligation- and transposon-based methods. Conclusions Various analytical factors were evaluated, and the pipeline of targeted gene sequencing using LR-PCR and MPS was established. These data can enable the optimization of targeted gene sequencing using LR-PCR and MPS in the clinical laboratory.

Anti‐PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c‐myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large‐scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti‐FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti‐FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti‐FIRΔexon2 Ab had higher sensitivity than anti‐FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti‐FIRΔexon2 Ab as candidate markers such as anti‐p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti‐FIRΔexon2 Ab and anti‐p53 Ab. In summary, our results suggest the use of anti‐FIRΔexon2 Ab in combination with the anti‐p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti‐FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.

Surgical site infections (SSIs) are significant and frequent perioperative complications, occurring due to the contamination of the surgical site. The late detection of SSIs, especially organ/space SSIs which are the more difficult to treat, often leads to severe complications. An effective method that can identify bacteria with a high accuracy, leading to the early detection of organ/space SSIs, is needed. Ninety-eight drainage fluid samples obtained from 22 patients with hepatobiliary pancreatic disease were analyzed to identify microorganisms using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) with a new membrane filtration protocol and rapid BACpro® pretreatment compared to sole rapid BACpro® pretreatment. The levels of detail of rapid BACpro® pretreatment with or without filtration were also evaluated for the accuracy of bacterial identification. We found that reliable scores for E. coli and E. faecalis were obtained by inoculation with 1.0 × 104 CFU/ml after preparation of the membrane filter with rapid BACpro®, indicating approximately 10-folds more sensitive compared to sole rapid BACpro® pretreatment in drainage fluid specimens. Among 60 bacterial positive colonies in drainage fluid specimens, the MALDI-TOF MS and the membrane filtration with rapid BACpro® identified 53 isolates (88.3%) with a significantly higher accuracy, compared to 25 isolates in the rapid BACpro® pretreatment group (41.7%) (p < 0.001). Among the 78 strains, 14 enteric Gram-negative bacteria (93.0%) and 55 Gram-positive cocci (87.3%) were correctly identified by the membrane filtration with rapid BACpro® with a high reliability. This novel protocol could identify bacterial species within 30 min, at $2-$3 per sample, thus leading to cost and time savings. MALDI-TOF MS with membrane filter and rapid BACpro® is a quick and reliable method for bacterial identification in drainage fluids. The shortened analysis time will enable earlier selection of suitable antibiotics for treatment of organ/space SSIs to improve patients’ outcomes.

Background: Atherosclerosis has been considered as the main cause of morbidity, mortality, premature incapacity, and disability worldwide. For early and sensitive diagnosis, development of novel biomarkers is expected and of significant practical importance.Methods: The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning (SEREX). Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen.Results: SEREX screening has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. As per the results of AlphaLISA, it was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke (AIS), transient ischemic attack (TIA), diabetes mellitus (DM), cardiovascular disease (CVD), chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were noted to be higher than that of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The Japan Public Health Center-based Prospective Study results have verified that the serum antibody levels against AP3D1 showed significantly higher odds ratios with the risk of AIS for persons with the third quartiles and the highest quartiles versus the lowest quartile, indicating that this antibody marker is deemed useful as risk factors for AIS.Conclusions: Serum anti-AP3D1 antibodies, which are broad-spectrum biomarkers of atherosclerotic diseases and digestive organ cancers, could be useful in predicting the onset of AIS.

C4b-binding protein α-chain (C4BPA) was previously identified as a novel serum biomarker for pancreatic ductal adenocarcinoma (PDAC). To apply this biomarker for clinical diagnosis, a lectin ELISA was established to measure serum fucosylated (Fuc)-C4BPA levels in 45 patients with PDAC, 20 patients with chronic pancreatitis (CP) and 50 healthy volunteers (HVs) in one training and three validation sets. The lecithin ELISA developed in the current study exhibited satisfactory within-run (2.6-6.7%) and between-day (1.8-3.6%) coefficient of variations. Serum Fuc-C4BPA levels in patients with PDAC (0.54±0.27 AU/ml) was significantly higher than that in HVs (0.21±0.06 AU/ml; P

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 μg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.

Background The most successful application of mass spectrometry (MS) in laboratory medicine is identification (ID) of microorganisms using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in blood stream infection. We describe MALDI-TOF MS-based bacterial ID with particular emphasis on the methods so far developed to directly identify microorganisms from positive blood culture bottles with MALDI-TOF MS including our own protocols. We touch upon the increasing roles of Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) as well. Main body Because blood culture bottles contain a variety of nonbacterial proteins that may interfere with analysis and interpretation, appropriate pretreatments are prerequisites for successful ID. Pretreatments include purification of bacterial pellets and short-term subcultures to form microcolonies prior to MALDI-TOF MS analysis. Three commercial protocols are currently available: the Sepsityper® kit (Bruker Daltonics), the Vitek MS blood culture kit (bioMerieux, Inc.), and the rapid BACpro® II kit (Nittobo Medical Co., Tokyo). Because these commercially available kits are costly and bacterial ID rates using these kits are not satisfactory, particularly for Gram-positive bacteria, various home-brew protocols have been developed: 1. Stepwise differential sedimentation of blood cells and microorganisms, 2. Combination of centrifugation and lysis procedures, 3. Lysis-vacuum filtration, and 4. Centrifugation and membrane filtration technique (CMFT). We prospectively evaluated the performance of this CMFT protocol compared with that of Sepsityper® using 170 monomicrobial positive blood cultures. Although preliminary, the performance of the CMFT was significantly better than that of Sepsityper®, particularly for Gram-positive isolates. MALDI-TOF MS-based testing of polymicrobial blood specimens, however, is still challenging. Also, its contribution to assessment of susceptibility and resistance to antibiotics is still limited. For this purpose, liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) should be more useful because this approach can identify as many as several thousand peptide sequences. Conclusion MALDI-TOF MS is now an essential tool for rapid bacterial ID of pathogens that cause blood stream infection. For the purpose of assessment of susceptibility and resistance to antibiotics of the pathogens, the roles of liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) will increase in the future.

Brahma-related gene 1 (BRG1), an ATPase subunit of the SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex controls multipotent neural crest formation by regulating epithelial-mesenchymal transition (EMT)-related genes with adenosine triphosphate-dependent chromodomain-helicase DNA-binding protein 7 (CHD7). The expression of BRG1 engages in pre-mRNA splicing through interacting RNPs in cancers; however, the detailed molecular pathology of how BRG1and CHD7 relate to cancer development remains largely unveiled. This study demonstrated novel post-transcriptional regulation of BRG1 in EMT and relationship with FIRΔexon2, which is a splicing variant of the far-upstream element-binding protein (FUBP) 1-interacting repressor (FIR) lacking exon 2, which fails to repress c-myc transcription in cancers. Previously, we have reported that FIR complete knockout mice (FIR−/−) was embryonic lethal before E9.5, suggesting FIR is crucial for development. FIRΔexon2 acetylated H3K27 on promoter of BRG1 by CHIP-sequence and suppressed BRG1 expression post-transcriptionally; herein BRG1 suppressed Snai1 that is a transcriptional suppressor of E-cadherin that prevents cancer invasion and metastasis. Ribosomal proteins, hnRNPs, splicing-related factors, poly (A) binding proteins, mRNA-binding proteins, tRNA, DEAD box, and WD-repeat proteins were identified as co-immunoprecipitated proteins with FIR and FIRΔexon2 by redoing exhaustive mass spectrometry analysis. Furthermore, the effect of FIRΔexon2 on FGF8 mRNA splicing was examined as an indicator of neural development due to impaired CHD7 revealed in CHARGE syndrome. Expectedly, siRNA of FIRΔexon2 altered FGF8 pre-mRNA splicing, indicated close molecular interaction among FIRΔexon2, BRG1 and CHD7. FIRΔexon2 mRNA was elevated in human gastric cancers but not in non-invasive gastric tumors in FIR+/ mice (K19-Wnt1/C2mE x FIR+/−). The levels of FIR family (FIR, FIRΔexon2 and PUF60), BRG1, Snai1, FBW7, E-cadherin, c-Myc, cyclin-E, and SAP155 increased in the gastric tumors in FIR+/− mice compared to those expressed in wild-type mice. FIR family, Snai1, cyclin-E, BRG1, and c-Myc showed trends toward higher expression in larger tumors than in smaller tumors in Gan-mice (K19-Wnt1/C2mE). The expressions of BRG1 and Snai1 were positively correlated in the gastric tumors of the Gan-mice. Finally, BRG1 is a candidate substrate of F-box and WD-repeat domain-containing 7 (FBW7) revealed by three-dimensional crystal structure analysis that the U2AF-homology motif (UHM) of FIRΔexon2 interacted with tryptophan-425 and asparate-399 (WD)-like motif in the degron pocket of FBW7 as a UHM-ligand motif. Together, FIRΔexon2 engages in multi-step post-transcriptional regulation of BRG1, affecting EMT through the BRG1/Snai1/E-cadherin pathway and promoting tumor proliferation and invasion of gastric cancers.

Rationale 25-Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson-type reagent, 1,2,4-triazoline-3,5-dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson-type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. Methods Caged 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) analogues were prepared from 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione, DAP-PA) was used for LC/MS/MS analyses of VD metabolites. Results The solution stability of DAP-PA in ethyl acetate dramatically improved compared with that of the non-caged one. We measured the thermal retro-Diels-Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature-dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. Conclusions DAP-PA was stable enough for mid- to long-term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P 1.7 and P 2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P 1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification .

Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.

A 64-year-old man was admitted with acute onset disturbed consciousness. Cerebrospinal fluid analysis revealed pleocytosis and elevated protein, with negative cultures and PCR. Serum antibodies for autoimmune encephalitis were also negative. Brain magnetic resonance imaging (MRI) was unremarkable, but whole-body CT scan showed a tumor in the left lower lung lobe. Bronchial brush cytology demonstrated clusters of malignant cells, and 18 F-fluorodeoxyglucose positron emission tomography showed multiple lesions and increased uptake in the lung tumor. Clinically the patient had a stage IV lung carcinoma, graded as T3N3M1b (OSS). Steroid therapy had limited efficacy, but chemotherapy dramatically improved his neurological symptoms. Therefore, he was diagnosed with paraneoplastic autoimmune encephalitis based on the diagnostic criteria for paraneoplastic neurological syndromes. He died due to disease progression 14 months later. Subsequent postmortem examination revealed white ill-defined nodules in the left lung, with similar nodules in other organs. The brain weighed 1500 g before fixation, and a nodule was observed in the right precentral gyrus. Microscopically, the lung tumor was a pleomorphic carcinoma with an adenocarcinoma component. Multiple areas of micro-softening (≤500 μm) were identified in the cerebral cortex, gray-white matter junction and basal ganglia, and were distributed diffusely in both the limbic and non-limbic systems. Mild lymphocytic infiltrates were observed involving few intraparenchymal vessels. Few tumor metastases were observed in the right precentral gyrus. The multiple micro-softenings may reflect a chronic neuropathologic change of paraneoplastic autoimmune encephalitis. They were too small to be detected by brain MRI. However, these lesions may have the potential to cause the neurological symptoms in the acute phase because they were observed in many anatomical regions. We should pay attention to subtle findings such as micro-softenings when estimating the neuropathology of autoimmune encephalitis. Further investigations are needed to understand the characteristic neuropathology of this condition.

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

Background To assess the vitamin D nutritional status, serum total 25-hydroxyvitamin D (25(OH)D) concentration is measured. We used six automated 25(OH)D immunoassays (AIAs) available in Japan and certified by the Vitamin D Standardization Program (VDSP) at the U.S. Center for Disease Control and Prevention to assess the concordance of the assay results. Methods Serum total 25(OH)D concentrations in SRM 972a and 20 serum samples from patients were determined using three liquid chromatography-tandem mass spectrometry (LC-MS/MS) and six AIAs (pilot study), and an additional 110 serum samples were assessed by the six AIAs (surveillance study). The assay bias from the results of LC-MS/MS by Chiba University or consensus values (i.e. average of six AIAs) was estimated using the procedure described in CLSI document EP09-A3. Results LC-MS/MS at Chiba University could completely separate 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3, and the observed values including total 25(OH)D in SRM 972a were all within ±1·SD of the assigned values. All AIAs produced results greater than ±3·SD. In the pilot study, four of the six AIAs had an average percentage bias, as estimated by confidence interval (CI), larger than ±5% (acceptance criterion in CLSI); the bias converged from −6.5% to 3.2% after adjustment by LC-MS/MS. In the surveillance study, 25(OH)D concentrations in AIAs all adjusted to LC-MS/MS converged within ±5% from consensus values. However, some AIAs showed negative or positive bias from the consensus values. Conclusions Current AIAs in Japan continue to lack standardization. Manufacturers should implement quality assurance strategies so that their values more closely align to those of standard reference material 972a.

The present study was planned to identify novel serum antibody markers for digestive organ cancers. We have used screening by phage expression cloning and identified novel fourteen antigens in this experiment. The presence of auto-antibodies against these antigens in serum specimens was confirmed by western blotting. As for auto-antibodies against fourteen antigens, AlphaLISA (amplified luminescence proximity homogeneous assay) assay was performed in the sera of gastrointestinal cancers patients to confirm the results. Serum antibody levels against these fourteen recombinant proteins as antigens between healthy donors (HD) and esophageal squamous cell carcinoma (ESCC) patients, gastric cancer (GC), or colon cancer (CC) were compared. The serum levels of all fourteen auto-antibodies were significantly higher in ESCC and GC than those of HD. Among those auto-antibodies, except ECSA2 and CCNL2, were also detected significantly higher levels in CC than those of HD. Receiver operating curve (ROC) revealed similar results except CCNL2 in CC. AUC values calculated by ROC were higher than 0.7 in auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, HS3ST1, TUBA1B, TACSTD2, AKR1C3, BAMBI, DCAF15 in ESCC, auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, TACSTD2, AKR1C3, BAMBI, DCAF15 in GC, and auto-antibodies against TPI1, HOOK2, PUF60 in CC. AUC of the combination of HOOK2 and anti-p53 antibodies in ESCC was observed to be as high as 0.8228. Higher serum antibody levels against ten antigens could be potential diagnostic tool for ESCC. Higher serum antibody levels against eight antigens could be potential diagnostic tool for GC, and serum antibody levels against three antigens could be potential diagnostic tool for CC.

Purpose: The response rate of ifosfamide (IFM) monotherapy for small-cell lung cancer (SCLC) is reported as 42.4% in Japanese package insert. However, these efficacy data are based on clinical studies conducted in 1970s. This phase II study evaluated the efficacy and safety of IFM combination with recommended current supportive therapy for recurrent SCLC in second-line and heavily treated setting. Methods: Recurrent SCLC patients pretreated with one to three prior regimens received IFM monotherapy (1.5 g/m2 for 3 days every 3 weeks). Treatment was continued until disease progression or unacceptable toxicity. The primary end point was objective response rate. Results: Twelve patients were enrolled in the study from June 2009 to January 2013. The study was early terminated at interim analysis due to futility stop. Patient characteristics were as follows: median age was 65 years, 11 were males (91.7%) and eight (66.7%) and four (33.3%) were Performance Status 0 and 1, respectively. Four patients (33.3%) enrolled in second-line setting were all refractory relapse SCLC and 8 (66.7%) were heavily treated patients. No patient showed objective response. Stable disease was observed in 3 patients. Median progression-free survival and overall survival were 0.9 months (95% CI, 0.3–1.5) and 4.8 months (95% CI, 1.6–9.9), respectively. Although one grade 4 amylase increase possibly related to IFM was observed, toxicity profile was totally favorable. Conclusions: IFM monotherapy should not be used for refractory relapse or heavily treated SCLC, and no further investigation is required in these populations., ファイル公開：2019/02/01

Background and aims Direct measurement of arginine vasopressin (AVP) via immunoassays is not widely conducted, mainly because of technical constraints. Liquid chromatography–tandem mass spectrometry (LC/MS/MS) has been widely used as the gold standard in clinical chemistry. Here, we aimed to develop an MS-based assay to determine human plasma AVP and compare the results with those obtained using a conventional immunoassay. Materials and methods We developed a protocol using triple quadrupole MS coupled with LC for the measurement of human plasma AVP. Analytical evaluations of the method were performed, and the results obtained using LC/MS/MS and radioimmunoassay (RIA) were compared. Results The lower limit of quantification (LLOQ) for plasma AVP obtained using LC/MS/MS and RIA were 0.2 and 0.4 pg/mL, respectively. Although there was a weak overall correlation between the results obtained using the two different methods, the RIA results did not agree with the LC/MS/MS results, particularly at low concentrations. Conclusions AVP detection through RIA is not satisfactory compared with that using LC/MS/MS. Diagnostic values of direct AVP measurements must be evaluated based on the results obtained via sensitive and accurate MS-based methods rather than those obtained through RIA.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10-20%, 30-40%, and 50-60%). As a result, higher identification scores were obtained at 30-40% humidity than at 10-20% or 50-60% humidity. At 30-40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30-40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.

Aims/Introduction Impaired glucose tolerance (IGT) is a subtype of prediabetes, a condition having high risk for development to diabetes mellitus, but its pathophysiology is not fully understood. In the present study, we examined metabolic changes in IGT by using two types (D‐glucose [Glc] and partial hydrolysate of starch [PHS]) of oral glucose tolerance tests (OGTTs), with emphasis on serum incretins and metabolites. Materials and Methods We carried out the two types of OGTT (Glc/OGTT and PHS/OGTT) in 99 young Japanese individuals who had tested either positive (GU +; n = 48) or negative (GU −; n = 51) for glycosuria. After OGTT, they were sub‐grouped into five categories: normal glucose tolerance (NGT) in the GU − group (GU −/NGT; n = 49), NGT in the GU + group (GU +/NGT; n = 28), IGT (n = 12), diabetes mellitus (n = 1) and renal glycosuria (n = 9). Serum incretin and metabolites of GU −/NGT and IGT were then measured. Results When the serum insulin level at each time‐point during PHS/OGTT was expressed as its ratio relative to Glc/OGTT, it was increased time‐dependently in GU −/NGT, but not in IGT. Such an increase in the ratio was also detected of serum incretin levels in GU −/NGT, but not in IGT, suggesting a lack of deceleration of oligosaccharide absorption in IGT. Metabolome analysis showed a difference in the serum levels of two metabolites of unknown function in mammals, methylcysteine and sedoheptulose 1,7‐bisphosphate, between GU −/NGT and IGT. Conclusions Comparison of PHS/OGTT and Glc/OGTT showed that oligosaccharide absorption was accelerated in IGT. Methylcysteine and sedoheptulose 1,7‐bisphosphate could be novel markers for dysregulated glucose metabolism.