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Ideberg type III glenoid fractures are relatively rare, and the mechanisms of injury and treatment strategies remain controversial. We reviewed the fracture patterns and final radiological findings in four cases. All fractures were wider on the medial side than on the side of the articular surface. All patients had multiple ipsilateral rib fractures; three out of four patients had a concomitant acromion fracture. Therefore, the mechanism of injury for the fracture is assumed to be external force applied in the caudal direction to the medial side of the body of the scapula. The superior shoulder suspensory complex was disrupted in most cases. A good clinical outcome was obtained in two patients with percutaneous screw fixation for the glenoid fracture and rigid plate fixation for the acromion fracture and in one patient who did not have an acromion fracture. However, one patient who did not undergo fixation of the acromion fracture developed osteoarthrosis of the shoulder joint. In this type of fracture, it is important to stabilize the acromion fracture at the same time as fixation of the glenoid fracture.

Introduction: Nonampullary duodenal epithelial tumors are rare, but their prevalence is increasing. Various gastrointestinal cancers have been associated with microbiomes. We evaluated the characteristics of the salivary and duodenal microbiomes of patients with nonampullary duodenal epithelial tumors., Methods: Saliva and biopsy samples from the duodenal bulb and descending portion were obtained from 15 patients with nonampullary duodenal epithelial tumors and 10 controls. Next-generation sequencing was performed to identify bacteria for comparison., Results: Saliva samples had higher Amplicon Sequence Variants (ASVs) and more observed species than duodenal samples. Saliva samples from patients with nonampullary duodenal epithelial tumor were dominated by Bacteroidetes and Prevotella, whereas Proteobacteria and Neisseria were dominant in the control samples. The relative abundance of bacteria was higher in patients with nonampullary duodenal epithelial tumors. Most bacteria were classified as bacteria of oral origin. Oribacterium and Stomatobaculum were significantly higher in the saliva, duodenal bulb, and descending portion of patients with nonampullary duodenal epithelial tumors., Conclusion: Patients with nonampullary duodenal epithelial tumors had different salivary and duodenal microbiomes than controls. Bacteria types differed between groups at each site, and most bacteria of oral origin were more abundant in patients with nonampullary duodenal epithelial tumors., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare., (Copyright © 2024 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Flow imaging (FI) has emerged as a powerful tool to evaluate insoluble particles derived from protein aggregates as an orthogonal method to light obscuration (LO). However, few reports directly compare the FI and LO method in the size and number of protein particles in commercially available therapeutic protein injections. In this study, we measured the number of insoluble particles in several therapeutic protein injections using both FI and LO, and characterized these particles to compare the analytical performance of the methods. The particle counts measured using FI were much higher than those measured using LO, and the difference depended on the products or features of particles. Some products contained a large number of transparent and elongated particles, which could escape detection using LO. Our results also suggested that the LO method underestimates the size and number of silicone oil droplets in prefilled syringe products compared to the FI method. The count of particles ≥10 μm in size in one product measured using FI exceeded the criteria (6000 counts per container) defined in the compendial particulate matter test using the LO method. Thus precaution should be taken when setting the acceptance criteria of specification tests using the FI method., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) treatment remains a big challenge in the field of oncology. The liver disease (viral or not viral) underlying HCC turned out to be crucial in determining the biologic behavior of the tumor, including its response to treatment. The aim of this analysis was to investigate the role of the etiology of the underlying liver disease in survival outcomes., Patients and Methods: We conducted a multicenter retrospective study on a large cohort of patients treated with lenvatinib as first-line therapy for advanced HCC from both Eastern and Western institutions. Univariate and multivariate analyses were performed., Results: Among the 1232 lenvatinib-treated HCC patients, 453 (36.8%) were hepatitis C virus positive, 268 hepatitis B virus positive (21.8%), 236 nonalcoholic steatohepatitis (NASH) correlate (19.2%) and 275 had other etiologies (22.3%). The median progression-free survival (mPFS) was 6.2 months [95% confidence interval (CI) 5.9-6.7 months] and the median overall survival (mOS) was 15.8 months (95% CI 14.9-17.2 months). In the univariate analysis for OS NASH-HCC was associated with longer mOS [22.2 versus 15.1 months; hazard ratio (HR) 0.69; 95% CI 0.56-0.85; P = 0.0006]. In the univariate analysis for PFS NASH-HCC was associated with longer mPFS (7.5 versus 6.5 months; HR 0.84; 95% CI 0.71-0.99; P = 0.0436). The multivariate analysis confirmed NASH-HCC (HR 0.64; 95% CI 0.48-0.86; P = 0.0028) as an independent prognostic factor for OS, along with albumin-bilirubin (ALBI) grade, extrahepatic spread, neutrophil-to-lymphocyte ratio, portal vein thrombosis, Eastern Cooperative Oncology Group (ECOG) performance status and alpha-fetoprotein. An interaction test was performed between sorafenib and lenvatinib cohorts and the results highlighted the positive predictive role of NASH in favor of the lenvatinib arm (P = 0.0047)., Conclusion: NASH has been identified as an independent prognostic factor in a large cohort of patients with advanced HCC treated with lenvatinib, thereby suggesting the role of the etiology in the selection of patients for tyrosine kinase treatment. If validated, this result could provide new insights useful to improve the management of these patients., Competing Interests: Disclosure AC-G has received grants and personal fees from MSD, Eisai and Bayer; and is an adviser for MSD, Eisai, Bayer, Bristol-Myers Squibb, AstraZeneca and GSK. MK has received grants from Taiho Pharmaceuticals, Chugai Pharmaceuticals, Otsuka, Takeda, Sumitomo Dainippon-Sumitomo, Daiichi Sankyo, AbbVie, Astellas Pharma and Bristol-Myers Squibb; has received grants and personal fees from MSD, Eisai and Bayer; and is an adviser for MSD, Eisai, Bayer, Bristol-Myers Squibb, Eli Lilly and ONO Pharmaceutical. AJ reports personal fees from Eisai. All other authors have declared no conflicts of interest. Data sharing Data available on request from the authors., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Adenoid cystic carcinoma (AdCC) is a rare, indolent salivary gland tumor that is reported to be driven by fusion genes. However, MYB/MYBL1-NFIB fusions have been detected in <60% of all AdCC cases and the oncogenic driver mutations in approximately 40% of AdCC remain unknown. Our aim was to identify novel gene fusions in AdCC., Study Design: We investigated 20 AdCC cases using a targeted RNA sequencing panel to identify gene fusions and performed quantitative real-time reverse transcription polymerase chain reaction to assess MYB, MYBL1, and NFIB expression levels., Results: A total of 36 fusion transcripts in 15 cases were detected and validated by Sanger sequencing. The MYB-NFIB and MYBL1-NFIB fusion genes were detected in 9 and 3 cases, respectively, in a mutually exclusive manner. Furthermore, novel gene fusions, namely, NFIB-EPB41L2, MAP7-NFIB, NFIB-MCMDC2, MYBL1-C8orf34, C8orf34-NFIB, and NFIB-CASC20, were identified. Among them, NFIB-EPB41L2 and NFIB-MCMDC2 are thought to activate MYB and MYBL1 expression, respectively, through the insertion of a genomic segment in proximity to MYB and MYBL1 genes, respectively., Conclusion: Six novel gene fusions other than MYB/MYBL1-NFIB were identified. The detection of novel fusion genes and investigation of the molecular mechanism will contribute to the development of novel molecular targeted therapies for this disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aim: Intractable or persistent hiccups and nausea (IHN) are rarely associated with herpes zoster (HZ-IHN). We aimed to identify the clinical characteristics of HZ-IHN by comparing them with those of neuromyelitis optica spectrum disorder associated with IHN (NMOSD-IHN)., Methods: We collected 8 patients with HZ-IHN and 12 patients with NMOSD-IHN diagnosed between 2002 and 2020 from medical databases. Medical records including clinical information, laboratory data on serum anti-aquaporin 4 (AQP4) antibodies, serological or cerebrospinal fluid findings for the varicella zoster virus, medullary MRI findings, and efficacy of intravenous methylprednisolone pulse (IVMP) therapy were analyzed retrospectively., Results: The age of onset (69 ± 13 years versus 46 ± 17 years, P = 0.003), percentage of men [7/8 patients (88%) versus 3/12 patients (25%), P = 0.020], serum CRP levels (1.41 ± 1.17 mg/dL versus 0.14 ± 0.33 mg/dL, P = 0.018), and frequency of hemi-cranial nerve involvement [6/8 patients (75%) versus 1/12 patients (8%), P = 0.004] were significantly higher in patients with HZ-IHN than in those with NMOSD-IHN. The hypoglossal and vagus nerves were involved in 5/8 patients (63%) with HZ-IHN. Other clinical parameters, excluding anti-AQP4 antibodies, were similar to those of NMOSD-IHN. MRI revealed ipsilateral hemi-dorsal medullar hyper-intense lesions in 5/8 patients (63%) with HZ-IHN. Acyclovir with IVMP therapy was effective for HZ-IHN., Conclusion: Clinicians should include HZ-IHN in the differential diagnosis for IHN, and promptly administer acyclovir and IVMP therapy. HZ-IHN is frequently accompanied by lower hemi-cranial nerve palsies and ipsilateral hemi-dorsal medullary hyper-intensity on MRI., Data Available Statement: The authors confirm that the data supporting the findings of this study are available within the article (Tables 1 and 2), or its supplementary materials (Table S1)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: After the advent of new treatment options for advanced hepatocellular carcinoma (HCC), the identification of prognostic factors is crucial for the selection of the most appropriate therapy for each patient., Patients and Methods: With the aim to fill this gap, we applied recursive partitioning analysis (RPA) to a cohort of 404 patients treated with lenvatinib., Results: The application of RPA resulted in a classification based on five variables that originated a new prognostic score, the lenvatinib prognostic index (LEP) index, identifying three groups: low risk [patients with prognostic nutritional index (PNI) >43.3 and previous trans-arterial chemoembolization (TACE)]; medium risk [patients with PNI >43.3 but without previous TACE and patients with PNI <43.3, albumin-bilirubin (ALBI) grade 1 and Barcelona Clinic Liver Cancer stage B (BCLC-B)]; high risk [patients with PNI <43.3 and ALBI grade 2 and patients with PNI <43.3, albumin-bilirubin (ALBI) grade 1 and Barcelona Clinic Liver Cancer stage C (BCLC-C)]. Median overall survival was 29.8 months [95% confidence interval (CI) 22.8-29.8 months] in low risk patients (n = 128), 17.0 months (95% CI 15.0-24.0 months) in medium risk (n = 162) and 8.9 months (95% CI 8.0-10.7 months) in high risk (n = 114); low risk hazard ratio (HR) 1 (reference group), medium risk HR 1.95 (95% CI 1.38-2.74), high risk HR 4.84 (95% CI 3.16-7.43); P < 0.0001. The LEP index was validated in a cohort of 127 Italian patients treated with lenvatinib. While the same classification did not show a prognostic value in a cohort of 311 patients treated with sorafenib, we also show a possible predictive role in favor of lenvatinib in the low risk group., Conclusions: LEP index is a promising, easy-to-use tool that may be used to stratify patients undergoing systemic treatment of advanced HCC., Competing Interests: Disclosure The authors have declared no conflicts of interest. Data sharing Data are available upon reasonable request., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pre-filled syringes (PFS) have been in widespread use as an administration device for therapeutic antibodies in recent decades. Generally, the inner barrel and syringe of PFS are coated with silicone oil (SO) for lubrication. Multiple studies have focused on the fact that the SO adsorbs denatured antibody molecules, and induces antibody aggregation. Aggregated antibodies are recognized as a potential risk for evoking immunogenic responses in patients. The characteristics of the aggregated antibody-SO complexes, including their concentration, population, shape, three-dimensional (3D) image, and Fcγ Receptors (FcγRs) activation have been obscurely acknowledged so far. In the present work, we prepared aggregated antibody-SO complexes by agitation and analyzed using multifaceted techniques such as flow imaging, confocal fluorescence microscopy, and cell-based assays for FcγRs activation. The results emphasized that the SO accelerates the increase in sub-visible particles and antibody aggregation. The confocal fluorescence microscopy analysis revealed the high-resolution 3D images of aggregated antibody-SO complexes. The FcγRs reporter cell assay clarified that the pre-mixed and agitated Ab + SO have higher FcγRs activation capability compared to the agitated Ab. Overall, this study advances the view that SO has an effect to increase the risk of agitation-induced aggregated antibody particles., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The abundance and composition of anthropogenic marine debris from 2012 to 2014 was assessed according to three bottom trawl surveys conducted on the upper continental slope between 198 m and 501 m off the Pacific coast of northeastern Japan. The surveys quantitated marine debris as follows: 33.52-164.62 items km -2 (January to July 2012), 91.68-215.11 items km -2 (November 2012 to May 2013), and 160.13-178.19 items km -2 (November 2013 to May 2014). Plastic bags or household materials mainly dominated terrestrial sources of debris. Principal component analysis latitudinally divided the study area according to debris abundance caused by geographical and hydrodynamic features. The long-term effect of tsunami-associated debris on the seafloor environment was recognized, because terrestrial sources such as heavy household materials were most abundant throughout the study period, with the additional accumulation of fishing gear and plastic bags., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019. At the 2018 meeting, Dr. Rukman DeSilva of the U.S. Food and Drug Administration and Dr. Srivalli Telikepalli of the National Institute of Standards and Technology participated as guest speakers. At the 2019 meeting, we invited Prof. John Carpenter of the University of Colorado, Prof. Gerhard Winter and Prof. Wolfgang Friess of Ludwig Maximilian University of Munich, and Dr. Tim Menzen of Coriolis Pharma Research, as guest commentators. In both meetings, the main research topic was strategies for the characterization and control of protein aggregates/subvisible particles in drug products. Specifically, the use of the light obscuration method for insoluble particulate matter testing with reduced injection volumes, and a comparison of analytical performance between flow imaging and light obscuration were discussed. Other topics addressed included host cell protein analysis, bioassay, and quality control strategies. In this commentary, the recent achievements of the research group, meeting discussions, and future perspectives are summarized., (Copyright © 2020. Published by Elsevier Inc.)

Distal hereditary motor neuropathies (dHMNs) comprise a group of clinically and genetically heterogeneous inherited lower motor neuron syndromes mainly characterized by a distal-predominant pattern of progressive muscle atrophy, weakness and hyporeflexia, without sensory dysfunction. Although at least 21 causative genes for dHMN have been reported, mutational scanning of these genes often fails to identify the causative variants in dHMN cohorts, suggesting that additional causative genes remain to be identified. We studied a four-generation pedigree of a Japanese family with autosomal dominant dHMN to provide insight into the pathogenetic basis of the disease. Neurological examinations were performed on all six family members enrolled in this study. Whole-exome sequencing (WES) was used to identify the causative gene for dHMN. The clinical features of the patients included muscle weakness with distal extensor dominancy in the lower extremities, accompanied by facial and neck flexor muscle impairment, no sensory involvement, and areflexia. Nerve conduction studies demonstrated axonal changes mainly in the peroneal nerve. WES combined with rigorous filtering revealed three missense variants (NM&#95;001083964: c.851G > A [p.Arg284His] in TDRKH, NM&#95;002858: c.1654G > T [p.Gly552Cys] in ABCD3, NM&#95;001005164: c.898A > T [p.Ile300Phe], in OR52E2). The variant in TDRKH is located in a conserved region of the tudor domain which is also present in the survival of motor neuron (SMN) protein, encoded by the SMN1 gene. Therefore, we concluded the variant in TDRKH is likely to be responsible for dHMN in our pedigree., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

With the use of an in situ and static method for gamma-ray measurements, levels of radioactive cesium 137 on shallow rugged reefs which lie between 37.3° N and 37.4° N, from the coastline of Fukushima to 141.06° E, at a depth of around 10 m were surveyed for the first time from May 2016 to December 2017. To confirm the contact between the detector and a surface of rock, we used a fact that potassium containing minerals are abundant and uniformly distributed in the area, and thus the strength of the photoelectric peak of natural radioactive potassium 40 is nearly constant over the area. We have found that the levels of radioactive cesium 137 varied from point to point within a range from 1 × 10 4  Bq/m 2 to 6 × 10 4  Bq/m 2 ., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Spinocerebellar ataxia 27 (SCA27) is an autosomal dominant SCA caused by variants in the fibroblast growth factor 14 (FGF14) gene. We examined a Japanese SCA patient whose deceased father also suffered from SCA. The patient was a 63-year-old male. He graduated from junior high school but received no further education. The predominant complaint was slowly progressive dysarthria and gait disturbance, which appeared at age 47. He showed pathological saccadic dysmetria, saccadic intrusions into smooth pursuit eye movements, dysarthria, and limb and truncal ataxia. His gait was wide-based but he did not require a walking stick. Limb muscle strength was intact. Deep tendon reflexes were normal or slightly reduced. Pathological reflexes were absent. He demonstrated mildly impaired vibration sense in the lower limbs. There was no urinary dysfunction. Brain MRI showed cerebellar atrophy without brainstem involvement. We first confirmed the absence of repeat expansion in genes known to be responsible for SCAs 1-3, 6-8, 10, 12, 17, 36 and dentatorubral-pallidoluysian atrophy. By exome analysis, we identified a novel heterozygous variant (NM&#95;004115, c.529A>T; Lys177X) in exon 4 of the FGF14 gene. This variant is expected to generate a truncated FGF14 protein lacking the heparin binding sites, those are likely to modify the activity of FGF14. We confirmed the absence of the variant in 502 healthy Japanese individuals by Sanger sequencing. There is no record of the variant in public databases. We conclude that the novel variation in FGF14 is causative for SCA27 in this patient., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Air embolisms are serious complications during catheter ablation procedures., Objectives: The aims of the present study were to determine when air bubbles enter the left atrium (LA) during catheter ablation procedures and to identify techniques that reduce air bubble intrusion., Methods: An ex vivo study was performed to monitor air bubbles using a silicone heart model and a high-resolution camera. In total, 280 radiofrequency catheter and cryoballoon ablation processes were tested., Results: Small and large air bubbles were often observed during catheter ablation processes. Many small air bubbles arose during sheath flushing at fast speeds (15 mL/2 s) (median bubble number [quartiles]: 35 [20-53] for SL0, 35 [23-44] for Agilis, and 98 [91-100] for FlexCath) and during initial cryoballoon inflation/freezing/deflation (34 [22-47]). Large (≥1.5 mm) air bubbles were observed during Lasso catheter insertion (1 [0-1]), cryoballoon insertion (2 [1-2]), and initial inflation/freezing/deflation (1 [1-3]). Massive air bubbles were observed during Optima catheter insertion into the sheath using an inserter (10 [2-15]). Sheath flushing at slow speeds (15 mL/5 s) significantly reduced the number of air bubbles. Before cryoballoon insertion, temporary balloon inflation and air bubble removal from the inflated surface were most effective in reducing air bubble intrusions. Optima catheter insertion without an inserter significantly reduced large air bubble intrusion., Conclusion: Air bubbles entered the LA at specific times. Techniques such as sheath flushing at slow speeds, temporary cryoballoon inflation before insertion, inserting the Optima catheter without an inserter, and avoidance of negative pressure in the LA could reduce air bubble intrusion., (Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Light chains are abnormally overexpressed from disordered monoclonal B-cells and form amyloid fibrils, which are then deposited on the affected organ, leading to a form of systemic amyloidosis known as AL (Amyloid Light chain) amyloidosis. A green tea catechin, epigallocatechin-3-O-gallate (EGCG), which is thought to inhibit various amyloidoses, is a potent inhibitor of amyloid fibril formation in AL amyloidosis., Methods: An amyloidogenic variable domain in λ6 light chain mutant, Wil was incubated in the presence of EGCG. The incubation products were analyzed by SDS-PAGE and reverse-phase HPLC. The interaction between Wil and EGCG was observed by using NMR and tryptophan fluorescence., Results: EGCG inhibited the amyloid fibril formation of Wil at pH 7.5 and 42 °C. Under these conditions, most Wil populations were in the unfolded state and several chemical reactions, i.e., oxidation and/or covalent bond oligomerization could be induced by auto-oxidated EGCG. Moreover, we found that EGCG bound to the unfolded state of Wil with higher affinity (Kd = 7 μM)., Conclusions: Inhibition of amyloid fibril formation of Wil was caused by 1) EGCG binding to unfolded state rather than folded state and 2) chemical modifications of Wil by auto oxidation of EGCG., General Significance: In the competitive formation of amyloid fibrils and off-pathway oligomers, EGCG produces the latter immediately after it preferentially binds to the unfolded state. It may be general mechanism of EGCG inhibition for amyloidosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: To investigate optic disc margin anatomic features in myopic eyes with open-angle glaucoma (OAG) using spectral-domain (SD) OCT., Design: Cross-sectional study., Participants: Two hundred four eyes of 102 participants with OAG and 106 eyes of 53 participants without glaucoma with axial length of 24 mm or more., Methods: Radial SD OCT B-scans centered on the optic discs were acquired in each eye, and the SD OCT data were colocalized with the optic disc stereophotographs. Optic disc margin anatomic features were evaluated as (1) SD OCT structure coinciding with the disc margin identified in the stereophotograph, (2) border tissue configuration, and (3) presence of Bruch's membrane overhang, and their frequency was computed in each clock-hour position. Further, paired eyes of myopic participants with OAG were divided into eyes with better or worse visual field defect (VFD), according to the mean deviation of the Humphrey visual field test, and associated factors were compared., Main Outcome Measures: Spectral-domain OCT structures coinciding with the visible optic disc margin in stereophotographs., Results: In myopic eyes with OAG, mean axial length was 25.96±1.07 mm and mean deviation was -8.87±7.78 dB. In approximately 90% of the participants, anterior scleral opening (ASO) coincided with the temporal disc margin and Bruch's membrane opening (BMO) with the nasal disc margin. Border tissue configuration was externally oblique in the temporal region and internally oblique in the nasal region of the optic disc. Bruch's membrane overhang was observed in a relatively small percentage of eyes. The same pattern of disc margin anatomic features was observed in the myopic eyes without glaucoma. The myopic optic disc was shaped by the temporal shifting of the BMO from the ASO, and the extent of shifting was expressed as the width of γ zone parapapillary atrophy (PPA). The greater γ zone PPA width was associated significantly with the worse VFD between paired eyes., Conclusions: The myopic eyes with OAG exhibited characteristic optic disc margin anatomic features that was considered to be derived from myopic deformation of the eye. The greater γ zone PPA width may increase susceptibility to the glaucomatous stress., (Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: Recent studies suggested protruding thrombus and atheroma after stent placement could be a substrate for subsequent adverse ischemic events. Although protruded atherothrombotic burden can be assessed as intra-stent tissue (IST) by optical coherence tomography (OCT), the effects of potent antiplatelet therapy on the acute phase resolution of IST in patients with acute coronary syndrome (ACS) was unknown., Methods: Ninety-six consecutive ACS patients with multi-vessel disease were enrolled in this prospective registry. In combination with aspirin, either clopidogrel or prasugrel was selected according to the date of enrolment. OCT examination was done immediately after percutaneous coronary intervention (post-PCI) and 10 days after index PCI (follow-up acute phase) to calculate averaged IST score as semi-quantitative measures of IST. High residual platelet reactivity (HRPR) was defined as platelet reactivity units (PRU)≥240 by VerifyNow P2Y12 assay (Accumetrics Inc., San Diego, CA, USA)., Results: Thirty two patients (38 stents) were enrolled in the prasugrel group and sixty four patients (72 stents) in the clopidogrel group. Averaged IST scores post-PCI were similar between the two groups (0.68±0.41 vs. 0.68±0.40, p=0.99), which decreased in all of the prasugrel group and in 87.5% of the clopidogrel group (p=0.02). Consequently, changes in averaged IST score (delta averaged IST score) were significantly greater in the prasugrel group compared to those in the clopidogrel group (-0.411±0.288 vs. -0.299±0.270, p=0.045). The frequency of HRPR was significantly lower in the prasugrel group (10.0% vs 32.4%, p=0.028)., Conclusions: Prasugrel plus aspirin achieved greater acute phase reduction of IST than clopidogrel plus aspirin, which might underlie the clinical benefit of potent antiplatelet therapy in ACS. (UMIN000018751)., (Copyright © 2018. Published by Elsevier Ltd.)

This study aims to reveal spatial distribution of hydrogen sulfide and sulfur species in marine sediments in Hiroshima Bay, Japan, by direct analyses using a combination of detection tubes and X-ray absorption fine structure spectroscopy. In summer and autumn, the hydrogen sulfide concentration ranged from <0.1 to 4 mg-S L -1 . In this study, only hydrogen sulfide was observed in autumn and at two stations in summer. In contrast, some earlier studies reported in all seasons in Hiroshima Bay the presence of acid volatile sulfide, which is used as a proxy of sulfide content. The sulfur species in sediments were mainly identified as sulfate, thiosulfate, elemental sulfur, and pyrite. Thiosulfate was a minor component compared to the other sulfur species. The formation of pyrite and sulfur derived from hydrogen sulfide oxidation played an important role in the scavenging of hydrogen sulfide., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Since its founding in 1993 the International Long-term Ecological Research Network (ILTER) has gone through pronounced development phases. The current network comprises 44 active member LTER networks representing 700 LTER Sites and ~80 LTSER Platforms across all continents, active in the fields of ecosystem, critical zone and socio-ecological research. The critical challenges and most important achievements of the initial phase have now become state-of-the-art in networking for excellent science. At the same time increasing integration, accelerating technology, networking of resources and a strong pull for more socially relevant scientific information have been modifying the mission and goals of ILTER. This article provides a critical review of ILTER's mission, goals, development and impacts. Major characteristics, tools, services, partnerships and selected examples of relative strengths relevant for advancing ILTER are presented. We elaborate on the tradeoffs between the needs of the scientific community and stakeholder expectations. The embedding of ILTER in an increasingly collaborative landscape of global environmental observation and ecological research networks and infrastructures is also reflected by developments of pioneering regional and national LTER networks such as SAEON in South Africa, CERN/CEOBEX in China, TERN in Australia or eLTER RI in Europe. The primary role of ILTER is currently seen as a mechanism to investigate ecosystem structure, function, and services in response to a wide range of environmental forcings using long-term, place-based research. We suggest four main fields of activities and advancements for the next decade through development/delivery of a: (1) Global multi-disciplinary community of researchers and research institutes; (2) Strategic global framework and strong partnerships in ecosystem observation and research; (3) Global Research Infrastructure (GRI); and (4) a scientific knowledge factory for societally relevant information on sustainable use of natural resources., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Familial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D , we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening., (© 2018 by The American Society of Hematology.)

Krüppel-Like Factor 14 (KLF14) gene, which appears to be a master regulator of gene expression in the adipose tissue and have previously been associated with BMI and Type 2 diabetes (T2D) by large genome-wide association studies. In order to find predictive biomarkers for the development of T2D, it is necessary to take epigenomic changes affected by environmental factors into account. This study focuses on ageing and obesity, which are T2D risk factors, and examines epigenetic changes and inflammatory changes. We investigated DNA methylation changes in the Klf14 promoter region in different organs of mice for comparing aging and weight. We found that methylation levels of these sites were increased with aging and weight in the spleen, the adipose tissue, the kidney, the lung, the colon and the whole blood cells. In addition, in the spleen, the adipose tissue and the whole blood, these epigenetic changes were also significantly associated with inflammatory levels. Moreover, not only Klf14, but also expression levels of some downstream genes were decreased with methylation in the spleen, the adipose tissue and the whole blood cells. Taken together, our results suggest that methylation changes of Klf14 in those tissues may be associated with changes in gene expression and inflammation on the adipose tissue of obesity and T2D. In addition, the methylation changes in the whole blood cells may serve as a predictive epigenetic biomarker for the development of T2D., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MAP1B (microtubule-associated protein 1B) binds to microtubules and regulates microtubule dynamics. Previously, we showed calcium-dependent interaction between MAP1B and a calcium-binding protein ALG-2 (apoptosis-linked gene 2), which is involved in regulation of the protein secretion pathway. Although ALG-2 generally binds to proteins through two consensus binding motifs such as ABM-1 and ABM-2, the absence of these motifs in MAP1B suggests a unique binding mode between MAP1B and ALG-2. Here, we identified the region of mouse MAP1B responsible for binding to ALG-2, and found point mutations that abrogated binding of MAP1B to ALG-2. Furthermore, interaction between MAP1B and ALG-2 selectively prevented ALG-2 from binding to proteins with ABM-2 such as Sec31A, suggesting competition between MAP1B and ABM-2-containing proteins for binding to ALG-2. Consistently, in MAP1B knockout cells, co-localization of ALG-2 with Sec31A was increased. Moreover, overexpression of wild-type MAP1B, but not the MAP1B mutant defective in ALG-2 binding, altered localizations of ALG-2 and Sec31A into dispersed distributions, suggesting that MAP1B regulates localizations of ALG-2 and Sec31A in the cells. Finally, we found two cancer-associated mutations of human MAP1B located near ALG-2 binding sites. The introduction of the corresponding mutations in mouse MAP1B dramatically reduced the binding ability to ALG-2. Thus, these results suggest that MAP1B plays a role in regulation of ALG-2 and Sec31A localizations, and that dysregulation of calcium-dependent binding of ALG-2 to MAP1B might influence pathological conditions such as cancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015. Dose-dependent binding responses were observed for all nine anti-EPO antibodies in the anti-EPO panel by the SPR and BLI methods. In contrast, the ECL method did not clearly detect binding of low-affinity anti-EPO antibodies. Regarding IgG2 and IgM antibodies derived from the same clone, IgG2 exhibited a higher binding response in the SPR assay, whereas the IgM binding response was higher than that of IgG2 in the ECL assay. In the case of the BLI method, there was no consistent pattern observed in the binding responses of IgG2 or IgM. Results of the anti-EPO antibody reference panel, which contains a variety of monoclonal antibodies, indicated that the ability to detect ADAs differed among these assay methods. Therefore, with ligand-binding assays, differences in assay platforms can affect the sensitivity and other characteristics of assays to detect ADAs. These results show that understanding the analytical performance of ADA assays is important for an appropriate assessment of immunogenicity. Our study also indicated the benefits of using the established human ADA reference panel to assess the assay methods for ADA detection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: For the treatment of coronary bifurcation lesions, optimal guidewire (GW) recrossing after main vessel stenting is important for good stent apposition at the side branch (SB) orifice in kissing balloon inflation (KBI)., Methods: We analyzed 150 bifurcation lesions treated with single stenting following KBI in the three-dimensional optical coherence tomography (3D-OCT) bifurcation registry study (2015-16) and a single center experience (2012-16). OCT examination was performed after GW recrossing to the SB and after KBI. Patients were divided into two-dimensional (2D, n=78) and 3D groups (n=72) according to 2D- or 3D-OCT guidance. GW recrossing position, jailing configuration of the stent over the SB (divided into Link-connecting type: stent link connecting to the carina and Link-free type: no stent link at the carina) and stent apposition were compared between the groups., Results: Distal GW recrossing was achieved in 75.6% and 91.7% in the 2D and 3D groups, respectively (P=0.004). Compared with the 2D group, the incidence of incomplete stent apposition (ISA) toward the SB in the 3D group tended to be lower in the whole cohort (14.5±13.6% vs 10.0±9.0%, P=0.077), and was significantly lower in left main trunk bifurcations (18.7±12.8% vs 10.3±8.9%, P=0.014). Independent contributors to ISA were the Link-connecting type (β 0.089, P<0.001), distal GW recrossing (β -0.078, P=0.001), and age (β -0.0020, P=0.012)., Conclusion: Optimal GW recrossing under 3D-OCT guidance is feasible and improves stent apposition, which may lead to a better clinical outcome in the treatment of bifurcation lesions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Land use and climate changes affect on the economy and environment with different patterns and magnitudes in the watershed. This study used risk analysis model stochastic efficiency with respect to a function (SERF) to evaluate economic and environmental risks caused by four climate change scenarios (baseline, small-, mid- and large changes) and three land uses (paddy dominated, paddy-farmland mixture and farmland dominated for agriculture) in Teshio watershed in northern Hokkaido, Japan. Under the baseline climate conditions, the lower ranking of economic income of crop yield and higher ranking of pollutant load from agricultural land were both predicted in paddy dominated for agriculture, suggesting that the paddy dominated system caused higher risks of economic and environmental variables compared to other two land uses. Increase of temperature and precipitation increased crop yields under all three climate changes which resulted in increase of the ranking of economic income, indicating that those climate changes could reduce economic risk. The increased temperature and precipitation also accelerated mineralization of organic nutrient and nutrient leaching to river course of Teshio which resulted in increase of the ranking of pollutant load, suggesting that those climate changes could lead to more environmental risk. The rankings of economic income in mid- and large changes of climate were lower than that in small change of climate under paddy-farmland mixture and farmland dominated systems due to decrease of crop yield, suggesting that climate change led to more economic risk. In summary, the results suggested that increase in temperature and precipitation caused higher risks of both economic and environmental perspectives, and the impacts was higher than those of land use changes in the studied watershed. Those findings would help producers and watershed managers to measure the tradeoffs between environmental protection and agricultural economic development for making decision under land use and climate changes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The research and development of next-generation innovative medicines is a prominent interest of both the government and industries in Japan. On June 29, 2017, a kickoff meeting of a new research group focused on the quality issues of biopharmaceuticals was held in Tokyo with Prof. John Carpenter as an invited guest. The group's research focuses mainly on the evaluation and control of protein aggregates/subvisible particles in drug products, but the research topics also include glycan analysis, host-cell protein evaluation, bioassay validation, and analytical quality by design. The purpose of the group's activities is to resolve the critical and fundamental quality issues important to pharmaceutical companies through the collaboration of industries, academia, and regulatory agencies. In this commentary, our current plan to address these issues and the discussion at the kickoff meeting are described., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dominant intermediate Charcot-Marie-Tooth disease F (CMTDIF) is an autosomal dominant hereditary form of Charcot-Marie-Tooth disease (CMT) caused by variations in the guanine nucleotide-binding protein, subunit beta-4 gene (GNB4). We examined two Japanese familial cases with CMT. Case 1 was a 49-year-old male whose chief complaint was slowly progressive gait disturbance and limb dysesthesia that appeared at the age of 47. On neurological examination, he showed hyporeflexia or areflexia, distal limb muscle weakness, and distal sensory impairment with lower dominancy. Nerve conduction studies demonstrated demyelinating sensorimotor neuropathy with reduced action potentials in the lower limbs. Case 2 was an 80-year-old man, Case 1's father, who reported difficulty in riding a bicycle at the age of 76. On neurological examination, he showed areflexia in the upper and lower limbs. Distal sensory impairment in the lower limbs was also observed. Nerve conduction studies revealed mainly axonal involvement. Exome sequencing identified a novel heterozygous nonsynonymous variant (NM&#95;021629.3:c.659T > C [p.Gln220Arg]) in GNB4 exon 8, which is known to be responsible for CMT. Sanger sequencing confirmed that both patients are heterozygous for the variation, which causes an amino acid substitution, Gln220Arg, in the highly conserved region of the WD40 domain of GNB4. The frequency of this variant in the Exome Aggregation Consortium Database was 0.000008247, and we confirmed its absence in 502 Japanese control subjects. We conclude that this novel GNB4 variant is causative for CMTDIF in these patients, who represent the first record of the disease in the Japanese population., (Copyright © 2017. Published by Elsevier Masson SAS.)

Spastic paraplegia (SPG) type 28 is an autosomal recessive SPG caused by mutations in the DDHD1 gene. We examined a Japanese 54-years-old male patient with autosomal recessive SPG. His parents were consanguineous. He needed a wheelchair for transfer due to spastic paraplegia. There was a history of operations for bilateral hallux valgus, thoracic ossification of the yellow ligament, bilateral carpal tunnel syndrome, bilateral ankle contracture, and lumbar spinal canal stenosis. He noticed gait disturbance at age 14. He used a cane for walking in his 40s. On neurological examination, he showed hyperreflexia, spasticity, and weakness in the lower extremities and bilateral Babinski reflexes. Urinary dysfunctions and impaired vibration sense in the lower limbs were observed. By exome sequencing analysis using Agilent SureSelect and Illumina MiSeq, we identified 17,248 homozygous nucleotide variants in the patient. Through the examination of 48 candidate genes known to be responsible for autosomal recessive SPG, we identified a novel homozygous 4-bp deletion, c.914&#95;917delGTAA, p.Ser305Ilefs*2 in exon2 of the DDHD1 gene encoding phosphatidic acid-preferring phospholipase A1 (PA-PLA1). The mutation is expected to cause a frameshift generating a premature stop codon 3-bp downstream from the deletion. In consequence, the DDHD domain that is known to be critical for PLA1 activity is completely depleted in the mutated DDHD1 protein, predicted to be a functionally null mutation of the DDHD1 gene. By Sanger sequencing, we confirmed that both parents are heterozygous for the mutation. This variation was not detected in 474 Japanese control subjects as well as the data of the 1,000G Project. We conclude that the novel mutation in DDHD1 is the causative variant for the SPG28 patient that is the first record of the disease in Japanese population., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: This study aimed to assess chronic-phase suppression of neointimal proliferation and arterial healing following paclitaxel-coated (PTX) and bare metal stent (BMS) implantation in the superficial femoral artery using optical coherence tomography (OCT)., Methods: Twenty-five patients with 68 stents underwent an 8-month OCT follow-up. Besides standard OCT variables, neointimal characterization and frequencies of peri-strut low-intensity area (PLIA), macrophage accumulation, and in-stent thrombi were evaluated., Results: The mean neointimal thickness was significantly less with PTX stents (544.9±202.2 μm vs. 865.0±230.6 μm, p<0.0001). The covered and uncovered strut frequencies were significantly smaller and larger, respectively, in the PTX stent group vs. the BMS group (93.7% vs. 99.4%; p<0.0001, 4.0% vs. 0.4%; p<0.0001, respectively). Heterogeneous neointima was only observed in the PTX stent group (12.5% vs. 0%, p=0.017). The frequencies of PLIA and macrophage accumulation were significantly greater in the PTX stent group (87.2% vs. 67.6%, p=0.001 and 46% vs. 9.1%, p=0.003, respectively)., Conclusion: After 8 months, reduced neointimal proliferation was observed with PTX stent implantation. On the other hand, delayed arterial healing was observed compared with BMS., (Copyright © 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Diagnosis and treatment of bone metastasis requires various types of measures, specialists and caregivers. To provide better diagnosis and treatment, a multidisciplinary team approach is required. The members of this multidisciplinary team include doctors of primary cancers, radiologists, pathologists, orthopaedists, radiotherapists, clinical oncologists, palliative caregivers, rehabilitation doctors, dentists, nurses, pharmacists, physical therapists, occupational therapists, medical social workers, etc. Medical evidence was extracted from published articles describing meta-analyses or randomised controlled trials concerning patients with bone metastases mainly from 2003 to 2013, and a guideline was developed according to the Medical Information Network Distribution Service Handbook for Clinical Practice Guideline Development 2014. Multidisciplinary team meetings are helpful in diagnosis and treatment. Clinical benefits such as physical or psychological palliation obtained using the multidisciplinary team approaches are apparent. We established a guideline describing each specialty field, to improve understanding of the different fields among the specialists, who can further provide appropriate treatment, and to improve patients' outcomes., Competing Interests: S Kato, sponsored research (Chugai Pharmaceutical Co, Ltd); IO, honoraria (Taiho Pharmaceutical Co, Ltd); S Kinuya, data and safety monitoring board (Bayer Yakuhin, Ltd); NS, honoraria (Novartis Pharma); ST, honoraria (Astellas Pharma Inc, AstraZeneca plc, Daiichi Sankyo Co, Ltd, Novartis Pharma), sponsored research (Sanofi, Zenyaku Kogyo Co, Ltd, Taiho Pharmaceutical Co, Ltd, Boehringer Ingelheim Japan, Chugai Pharmaceutical Co, Ltd, Novartis Pharma); YT, honoraria (Janssen Pharma); K Takayama20, consultation and honoraria (Clinical Research Support Center Kyushu, AstraZeneca plc, Chugai Pharmaceutical Co, Ltd, Eli Lilly Japan, PfizerJapan Inc), sponsored research (Kyowa Hakko Kirin Co, Ltd, plc, Daiichi Sankyo Co, Ltd, Chugai Pharmaceutical Co, Ltd, Eli Lilly Japan, Novartis Pharma, Bristol-Myers Squibb); UT, consultation and honoraria (Micron Inc, SymBio Pharmaceutical Co, Ltd, Chugai Pharmaceutical Co, Ltd, Nihon Medi-Physics Co, Ltd); HM, sponsored research (Eisai Co, Ltd, Taiho Pharmaceutical Co, Ltd); T Yuasa, honoraria (Novartis Pharma, PfizerJapan Inc).

Protease-resistant, misfolded isoforms (PrP(Sc)) of a normal cellular prion protein (PrP(C)) in the bodily fluids, including blood, urine, and saliva, are expected to be useful diagnostic markers of prion diseases, and nonhuman primate models are suited for performing valid diagnostic tests for human Creutzfeldt-Jakob disease (CJD). We developed an effective amplification method for PrP(Sc) derived from macaques infected with the atypical L-type bovine spongiform encephalopathy (L-BSE) prion by using mouse brain homogenate as a substrate in the presence of polyanions and L-arginine ethylester. This method was highly sensitive and detected PrP(Sc) in infected brain homogenate diluted up to 10(10) by sequential amplification. This method in combination with PrP(Sc) precipitation by sodium phosphotungstic acid is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF), saliva, urine, and plasma of macaques that have been intracerebrally inoculated with the L-BSE prion. Furthermore, PrP(Sc) was detectable in the saliva or urine samples as well as CSF samples obtained at the preclinical phases of the disease. Thus, our novel method may be useful for furthering the understanding of bodily fluid leakage of PrP(Sc) in nonhuman primate models., (Copyright © 2016 Elsevier Inc. All rights reserved.)