Site-specific and linkage analyses of fucosylated N-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported observed in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 in only cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers

Dynamics of proteins with different molecular structures under solution condition

Incoherent quasielastic neutron scattering (iQENS) is a fascinating technique for investigating the internal dynamics of protein. However, low flux of neutron beam, low signal to noise ratio of QENS spectrometers and unavailability of well-established analyzing method have been obstacles for studying internal dynamics under physiological condition (in solution). The recent progress of neutron source and spectrometer provide the fine iQENS profile with high statistics and as well the progress of computational technique enable us to quantitatively reveal the internal dynamic from the obtained iQENS profile. The internal dynamics of two proteins, globular domain protein (GDP) and intrinsically disordered protein (IDP) in solution, were measured with the state-of-the art QENS spectrometer and then revealed with the newly developed analyzing method. It was clarified that the average relaxation rate of IDP was larger than that of GDP and the fraction of mobile H atoms of IDP was also much higher than that of GDP. Combined with the structural analysis and the calculation of solvent accessible surface area of amino acid residue, it was concluded that the internal dynamics were related to the highly solvent exposed amino acid residues depending upon protein’s structure

Administration of Bone Marrow-Derived Mononuclear Cells Contributed to the Reduction of Hypoxic-Ischemic Brain Injury in Neonatal Rats

Background/Objective: Perinatal hypoxic-ischemia (HI) causes neonatal death and permanent neurological deficits. Cell therapy using various cell sources has been recently identified as a novel therapy for perinatal HI. Among the available types of cell sources, bone marrow-derived mononuclear cells (BMMNCs) have unique features for clinical application. For example, stem cells can be collected after admission, thus enabling us to perform autologous transplantation. This study aimed to investigate whether the administration of BMMNCs ameliorated HI brain injury in a neonatal rat model.Methods: Seven-day-old rats underwent left carotid artery ligation and were exposed to 8% oxygen for 60 min. BMMNCs were collected from the femurs and tibias of juvenile rats using the Ficoll–Hypaque technique and injected intravenously 24 h after the insult (1 × 105 cells). Active caspase-3, as an apoptosis marker, and ED1, as an activated microglia/macrophage marker, were evaluated immunohistochemically 48 h after the insult (vehicle, n = 9; BMMNC, n = 10). Behavioral assessments using the rotarod treadmill, gait analysis, and active avoidance tests were initiated 3 weeks after the insult (sham, n = 9, vehicle, n = 8; BMMNC, n = 8). After these behavioral tests (6 weeks after the insult), we evaluated the volumes of their hippocampi, cortices, thalami, striata, and globus pallidus.Results: The mean cell densities of the sum of four parts that were positive for active caspase-3 significantly decreased in the BMMNC group (p < 0.05), whereas in the hippocampi, cortices, thalami, and striata cell densities decreased by 42, 60, 56, and 47%, respectively, although statistical significance was not attained. The number of ED1 positive cells for the sum of the four parts also significantly decreased in the BMMNC group compared to the vehicle group (p < 0.05), whereas in each of the four parts the decrease was 35, 39, 47, and 36%, respectively, although statistical significance was not attained. In gait analysis, the BMMNC normalized the contact area of the affected hind paw widened by HI. The volumes of the affected striata and globus pallidus were significantly larger in the BMMNC group than in the control group.Conclusion: These results indicated that the injection of BMMNCs ameliorated HI brain injury in a neonatal rat model

Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cell, but not Adipose Tissue-Derived Stem Cell, Ameliorated the Neonatal Hypoxic-Ischemic Brain Injury by Changing Cerebral Inflammatory State in Rat

Perinatal hypoxic-ischemic (HI) brain injury occurs in 1 in 1,000 live births and remains the main cause of neurological disability and death in term infants. Cytotherapy has recently emerged as a novel treatment for tissue injury. In particular, mesenchymal stem cells (MSCs) are thought to have therapeutic potential, but little is known about the differences according to their origin. In the current study, we investigated the therapeutic effects and safety of intravenous injection of allogeneic bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs) in a rat model of HI brain injury. HI models were generated by ligating the left carotid artery of postnatal day 7 Wistar/ST rats and exposing them to 8% hypoxia for 60 min. Bone marrow and adipose tissue were harvested from adult green fluorescent protein transgenic Wistar rats, and cells were isolated and cultured to develop BM-MSCs and ADSCs. At passaging stages 2–3, 1 × 105 cells were intravenously injected into the external right jugular vein of the HI rats at 4 or 24 h after hypoxia. Brain damage was evaluated by counting the number of cells positive for active caspase-3 in the entire dentate gyrus. Microglial isotypes and serum cytokines/chemokines were also evaluated. Distribution of each cell type after intravenous injection was investigated pathologically and bio-optically by ex vivo imaging (IVIS®) with a fluorescent lipophilic tracer DiR. The mortality rate was higher in the ADSC group compared to the BM-MSC group, in pups injected with cells 4 h after hypoxia. The number of active caspase-3-positive cells significantly decreased in the BM-MSC group, and the percentage of M1 microglia (a proinflammatory isotype) was also lower in the BM-MSC vs control group in the penumbra of the cortex. Moreover, BM-MSC administration increased anti-inflammatory cytokine and growth factor levels, while ADSCs did not. Each injected cell type was mainly distributed in the lungs and liver, but ADSCs remained in the lungs longer. Pathologically, pulmonary embolisms and diffuse alveolar hemorrhages were seen in the ADSC group. These results indicated that injection of allogeneic BM-MSCs ameliorated neonatal HI brain injury, whereas ADSCs induced severe lung hemorrhage and higher mortality

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133− Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133+ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133+ and CD133− colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10+ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133+ and CD133− subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133+ cells showed significantly greater tumor growth than CD133− cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133+ cells exhibited significantly more invasive behaviors than CD133− cells (P<0.001), especially in cocultures with CD10+ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10+ fibroblasts enhanced the tumor growth of CD133+ cells significantly more than CD10− fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133+ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10+ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submit- Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia; 23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇ ic´ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany; 26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada; 27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore, Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363–883 Korea (South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363–700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon, Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158–8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey 08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20–22 rue Henri et Gilberte Goudier 63200 RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry, Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California 94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District, Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave, Davis, California 95616; 70Horva´ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary; 72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg, Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tu¨ bingen, Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007 Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 24, 2019, and in revised form, August 26, 2019 Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677 ER: NISTmAb Glycosylation Interlaboratory Study 12 Molecular & Cellular Proteomics 19.1 Downloaded from https://www.mcponline.org by guest on January 20, 2020 ted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide communityderived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Molecular & Cellular Proteomics 19: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

エタン，プロパンおよびイソブタンの脱水素により析出した大量の炭素に覆われたアルミナ担持酸化ニッケルの触媒再生

Improvement in the dehydrogenations of ethane, propane, and isobutane over alumina-supported nickel oxides occurs together with the formation of large amounts of carbon deposits with time-on-stream, but catalyst activity is decreased with an additional increase in time-on-stream. This improvement in activity is due to metallic nickel formation that is highly dispersed over carbon nanotube-like deposits. However, the activity decreases as this highly dispersed metallic nickel is further covered with carbon deposits. We describe the use of oxygen treatment to regenerate the catalyst. Oxygen treatment to remove carbon deposits generally results in a less active catalyst due to sintering of the active species. However, we speculated that sintered nickel oxide could form carbon nanotubes in the proposed system, and that the formation of highly dispersed nickel over the nanotubes would regenerate the catalyst. To prove this hypothesis, dehydrogenations of ethane, propane, and isobutane were investigated using 18, 15, and 20 % nickel oxide supported on γ-alumina, respectively. We confirmed regeneration of the catalytic activity via oxygen treatment during subsequent dehydrogenations.アルミナ担持酸化ニッケル触媒によるエタン，プロパン，およびイソブタンの脱水素化では，通塔時間に伴う炭素析出の形成とともに，触媒活性の向上が観察されることが報告されている。この改善挙動はカーボンナノチューブ状析出物上に高分散状態で形成される金属ニッケルに起因されるが，さらに通塔時間を長くすると，カーボンナノチューブ状析出物が通常の炭素析出物によって覆われ，活性が低下する。本稿では，活性が低下したアルミナ担持酸化ニッケルを酸素処理により再生した結果について述べた。 炭素堆積物を除去するために酸素処理を用いると，活性種のシンタリングより，触媒活性成分が低分散化され，活性が低下することが一般に知られている。しかしながら，本触媒系では，酸素処理でシンタリングした低分散の酸化ニッケルが形成されたとしても，再度接触反応に用いると，そこからカーボンナノチューブが形成され，このナノチューブ上に高分散状態で金属ニッケルが形成され，良好な触媒活性が再生されることが期待される。この仮説を証明するために，エタン，プロパン，およびイソブタンの脱水素化を，γ-アルミナに酸化ニッケルを18 %，15 %，および 20 %担持した触媒を用いて検討した。その結果，これら3種類のアルカンの脱水素に対して，本稿で提案した酸素処理による触媒活性の再生が良好に行われることが明らかになった

積層型シート状酵素バイオ電池の構築

We have constructed a sheet-type glucose/dioxygen enzymatic biofuel cell with multi-stacked structure, in which one biocathode is sandwiched with two bioanodes in parallel. The energy density of the biofuel cell reached 14.1 mWh cm−3. The cell has achieved the acceptable performance on re-fueling, and the stability of the cell before use was drastically improved by adding raffinose

Data Collection for Dilute Protein Solutions via a Neutron Backscattering Spectrometer

Understanding protein functions requires not only static but also dynamic structural information. Incoherent quasi-elastic neutron scattering (QENS), which utilizes the highly incoherent scattering ability of hydrogen, is a powerful technique for revealing the dynamics of proteins in deuterium oxide (D2O) buffer solutions. The background scattering of sample cells suitable for aqueous protein solution samples, conducted with a neutron backscattering spectrometer, was evaluated. It was found that the scattering intensity of an aluminum sample cell coated with boehmite using D2O was lower than that of a sample cell coated with regular water (H2O). The D2O-Boehmite coated cell was used for the QENS measurement of a 0.8 wt.% aqueous solution of an intrinsically disordered protein in an intrinsically disordered region of a helicase-associated endonuclease for a fork-structured type of DNA. The cell was inert against aqueous samples at 283&ndash;363 K. In addition, meticulous attention to cells with small individual weight differences and the positional reproducibility of the sample cell relative to the spectrometer neutron beam position enabled the accurate subtraction of the scattering profiles of the D2O buffer and the sample container. Consequently, high-quality information on protein dynamics could be extracted from dilute protein solutions