Publications by authors named "Motoko Takahashi"

75 Publications

Feasibility and educational value of fluorescence cholangiography in laparoscopic cholecystectomy.

Asian J Endosc Surg 2021 Apr 5. Epub 2021 Apr 5.

Department of Digestive and General Surgery, Uonuma Kikan Hospital, Niigata, Japan.

Introduction: Near-infrared fluorescence cholangiography during a laparoscopic cholecystectomy has become widely accepted as a useful auxiliary tool to visualize the extrahepatic biliary structures. We investigated the feasibility and educational value of a method with longer interval between the administration of indocyanine green and the imaging of these structures.

Methods: Approximately 18 hours before their surgery, patients (n = 51) were intravenously administered 0.25 mg/kg of indocyanine green. Each laparoscopic cholecystectomy was performed under fluorescence imaging in combination with white-light imaging. Operative outcomes including visualization of the extrahepatic biliary structures and operative time were compared between the patients on whom board-certified surgeons operated (feasibility phase; n = 18) and the patients on whom a surgery resident operated (educational phase; n = 33).

Results: There were no adverse events related to the longer interval method. The visualization rates of extrahepatic biliary structures were comparable between the two phases. Both the mean time to divide the cystic duct and the mean time to remove the gallbladder in the educational phase were significantly longer than those in the feasibility phase (68.2 vs 24.4 minutes and 30.2 vs 15.8 minutes, P < .001 each). There was no significant difference in other operative outcomes. The operative time learning curve did not decrease with a resident's experience.

Conclusions: Fluorescence cholangiography with the longer interval method was feasible and could identify the extrahepatic biliary structures irrespective of the surgeon's experience; however, it did not decrease the operative time with experience.
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http://dx.doi.org/10.1111/ases.12939DOI Listing
April 2021

Treatment of air leakage using the VIO soft coagulation system: a mouse pulmonary air leak model.

Surg Today 2021 Mar 20. Epub 2021 Mar 20.

Department of Thoracic Surgery, School of Medicine and Hospital, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo, Hokkaido, 060-8543, Japan.

Purpose: We aimed to compare the efficacy of the VIO soft coagulation system (VSCS) for the treatment of air leaks by sealing with fibrin glue, and also assess the histological alterations that occur after soft coagulation.

Methods: A mouse pulmonary air leak model was designed. The pulmonary fistula was subsequently coagulated with the VSCS or sealed with fibrin glue with polyglycolic acid (PGA) sheets. The burst pressure at air leak recurrence was measured in each group, and the results were compared. We also evaluated the histological alterations in the mouse pulmonary air leak model after soft coagulation with the VSCS.

Results: The burst pressure in the soft coagulation group (80 W/Effect 5) (median 42.8; range 35.4-53.8 cmHO) was similar to that in the fibrin glue group (median 41.5; range 34.6-43.9 cmHO) (p = 0.21). Histological examinations revealed that the visceral pleura remained torn, the structure of the pulmonary alveolus was maintained, and the coagulated fistula was covered with a fibrin membrane in the soft coagulation group.

Conclusions: The pressure resistance following soft coagulation was equivalent to that after sealing using fibrin glue with PGA sheets. The air leaks were likely controlled by covering the fistula with a fibrin membrane after soft coagulation with the VSCS.
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http://dx.doi.org/10.1007/s00595-021-02251-3DOI Listing
March 2021

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation.

Curr Biol 2021 Feb 22. Epub 2021 Feb 22.

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan. Electronic address:

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.
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http://dx.doi.org/10.1016/j.cub.2021.01.062DOI Listing
February 2021

Retraction Note: Silencing of Glutathione S-Transferase Pi Inhibits Cancer Cell Growth via Oxidative Stress Induced by Mitochondria Dysfunction.

Sci Rep 2020 Nov 19;10(1):20589. Epub 2020 Nov 19.

Department of Probiotics Immunology, Institute for Genetic Medicine, Hokkaido University, N15W7, Sapporo, 001-0015, Japan.

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-77205-9.
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http://dx.doi.org/10.1038/s41598-020-77205-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678859PMC
November 2020

Acrolein in cigarette smoke attenuates the innate immune responses mediated by surfactant protein D.

Biochim Biophys Acta Gen Subj 2020 11 30;1864(11):129699. Epub 2020 Jul 30.

Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima, Hiroshima, Japan.

Background: Surfactant proteins (SP) A and D belong to collectin family proteins, which play important roles in innate immune response in the lung. We previously demonstrated that cigarette smoke (CS) increases the acrolein modification of SP-A, thereby impairing the innate immune abilities of this protein. In this study, we focused on the effects of CS and its component, acrolein, on the innate immunity role of another collectin, SP-D.

Methods: To determine whether aldehyde directly affects SP-D, we examined the lungs of mice exposed to CS for 1 week and detected aldehyde-modified SP-D using an aldehyde reactive probe. The structural changes in CS extract (CSE) or acrolein-exposed recombinant human (h)SP-D were determined by western blot, liquid chromatography-electrospray ionization tandem mass spectrometry, and blue native-polyacrylamide gel electrophoresis analyses. Innate immune functions of SP-D were determined by bacteria growth and macrophage phagocytosis.

Results: Aldehyde-modified SP-D as well as SP-A was detected in the lungs of mice exposed to CS for 1 week. Exposure of hSP-D to CSE or acrolein induced an increased higher-molecular -weight of hSP-D and acrolein induced modification of five lysine residues in hSP-D. These modifications led to disruption of the multimer structure of SP-D and attenuated its ability to inhibit bacterial growth and activate macrophage phagocytosis.

Conclusion: CS induced acrolein modification in SP-D, which in turn induced structural and functional defects in SP-D.

General Significance: These results suggest that CS-induced structural and functional defects in SP-D contribute to the dysfunction of innate immune responses in the lung following CS exposure.
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http://dx.doi.org/10.1016/j.bbagen.2020.129699DOI Listing
November 2020

Nuclear microenvironment in cancer: Control through liquid-liquid phase separation.

Cancer Sci 2020 Sep 21;111(9):3155-3163. Epub 2020 Jul 21.

Division of Cancer Biology, The Cancer Institute of JFCR, Tokyo, Japan.

The eukaryotic nucleus is not a homogenous single-spaced but a highly compartmentalized organelle, partitioned by various types of membraneless structures, including nucleoli, PML bodies, paraspeckles, DNA damage foci and RNA clouds. Over the past few decades, these nuclear structures have been implicated in biological reactions such as gene regulation and DNA damage response and repair, and are thought to provide "microenvironments," facilitating these reactions in the nucleus. Notably, an altered morphology of these nuclear structures is found in many cancers, which may relate to so-called "nuclear atypia" in histological examinations. While the diagnostic significance of nuclear atypia has been established, its nature has remained largely enigmatic and awaits characterization. Here, we review the emerging biophysical principles that govern biomolecular condensate assembly in the nucleus, namely, liquid-liquid phase separation (LLPS), to investigate the nature of the nuclear microenvironment. In the nucleus, LLPS is typically driven by multivalent interactions between proteins with intrinsically disordered regions, and is also facilitated by protein interaction with nucleic acids, including nuclear non-coding RNAs. Importantly, an altered LLPS leads to dysregulation of nuclear events and epigenetics, and often to tumorigenesis and tumor progression. We further note the possibility that LLPS could represent a new therapeutic target for cancer intervention.
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http://dx.doi.org/10.1111/cas.14551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469853PMC
September 2020

Insufficient serum L-ficolin is associated with disease presence and extent of pulmonary Mycobacterium avium complex disease.

Respir Res 2019 Oct 21;20(1):224. Epub 2019 Oct 21.

Department of Respiratory Medicine and Allergology, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo, 060-8556, Japan.

Background: The incidence of infectious disease caused by nontuberculous mycobacteria is increasing worldwide. Pulmonary Mycobacterium avium complex (MAC) disease is difficult to treat with chemotherapy, and its mechanism of infection, infection route, disease onset, and severity remain unknown. Ficolins are oligomeric defense lectins. L-ficolin plays an important role in innate immunity. This study's aim was to identify L-ficolin's role in patients with pulmonary MAC disease.

Methods: Between April 2011 and September 2017, 61 Japanese patients with pulmonary MAC disease were seen at our hospital. A control group, comprising 30 healthy individuals, without respiratory disease were enrolled in our study. The relationship between serum L-ficolin levels and disease severity was assessed, and L-ficolin's antibacterial role was examined.

Results: Serum L-ficolin levels were significantly lower in patients with pulmonary MAC disease than in healthy subjects (1.69 ± 1.27 μg/ml vs. 3.96 ± 1.42 μg/ml; p < 0.001). The cut-off value, based on receiver operating characteristic (ROC) analysis results, was 2.48 μg/ml (area under the curve (AUC) 0.90, sensitivity and specificity 83.6 and 86.7%, respectively). Serum L-ficolin levels were significantly lower in the patients with nodular bronchiectatic type disease compared with the patients with fibrocavitary type disease and were lower in the high-resolution computed tomography high-scoring group compared with low-scoring group. An in vitro analysis showed that purified recombinant L-ficolin bound to M. avium and its major cell wall component, lipoarabinomannan, in a concentration-dependent manner. In addition, recombinant L-ficolin suppressed M. avium growth in a concentration-dependent manner.

Conclusions: Insufficient serum L-ficolin is associated with disease progression in pulmonary MAC disease, and the level of serum L-ficolin is a possible biomarker.

Trial Registration: This study is registered with UMIN ( UMIN000022392 ).
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http://dx.doi.org/10.1186/s12931-019-1185-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805425PMC
October 2019

Silencing of Glutathione S-Transferase Pi Inhibits Cancer Cell Growth via Oxidative Stress Induced by Mitochondria Dysfunction.

Sci Rep 2019 10 14;9(1):14764. Epub 2019 Oct 14.

Department of Probiotics Immunology, Institute for Genetic Medicine, Hokkaido University, N15W7, Sapporo, 001-0015, Japan.

Antitumor drug development based on the concept of intervening in the antioxidant system of cancer cells has been gaining increased interest. In this study, we propose a promising strategy for cancer treatment using modulation of oxidative stress by suppression of glutathione S-transferases (GSTs), a typical antioxidant enzyme. siRNA which can be applied to the development of nucleic acid drugs, enabling them to eliminate unwanted side effects, increase specificity, and avoid the problem of drug resistance, was employed for GSTP-silencing at the transcriptional level. The silencing of the pi class of GST (GSTP) that displayed the most characteristic expression profile in 13 kinds of cancer cell lines has shown significant impairment in the growth of cancer cells due to oxidative stress caused by excess ROS accumulation. Comparative proteomics between normal cells and GSTP-silenced pancreatic cancer cell PANC-1 suggested that GSTP-silencing facilitated the mitochondrial dysfunction. These findings show promise for the development of strategies toward cancer therapy based on the mechanism that allows genetic silencing of GSTP to promote oxidative stress through mitochondria dysfunction.
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http://dx.doi.org/10.1038/s41598-019-51462-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791853PMC
October 2019

Folding the genome into mitotic chromosomes.

Curr Opin Cell Biol 2019 10 15;60:19-26. Epub 2019 Apr 15.

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Tokyo, Japan. Electronic address:

How linear DNA molecules are packaged into compact cylindrical chromosomes in preparation for cell division has remained one of the central outstanding questions in cell biology. Condensin is a highly conserved protein complex that universally determines large-scale DNA geometry during mitotic chromosome assembly. A wide range of recently developed approaches, including super resolution microscopy, single molecule imaging, Hi-C analyses and computational modeling, have profoundly changed how we view mitotic chromosomes. This review highlights recent discoveries on chromosome architecture and condensin function.
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http://dx.doi.org/10.1016/j.ceb.2019.03.005DOI Listing
October 2019

Comparison of immune response in mice sensitized to an animal allergen, Can f 1, and to a food allergen, ovalbumin.

Biomed Res 2019 ;40(1):9-15

Institute for Animal Experimentation, Hokkaido University.

Can f 1 belongs to the lipocalin superfamily and is considered to be an animal allergen. The immune response induced by Can f 1 in mice was compared with that induced by ovalbumin (OVA), a typical food allergen. Female BALB/c and C57BL/6 mice (6 weeks of age) were subcutaneously injected with Can f 1 or OVA with or without aluminum hydroxide (Alum) three times with intervals of two weeks. Serum levels of total IgE or antigen-specific IgE and production of IL13 and IFNγ from splenocytes were analyzed. Immunization with Can f 1 or OVA increased serum levels of both total IgE and antigen-specific IgE significantly irrespective of Alum. These results indicate that Can f 1 and OVA were able to induce allergic sensitization in mice. Splenocyte production of IL13 in mice immunized with Can f 1 or OVA with and without Alum were significantly increased after stimulation with each antigen. However, IL13 levels in the mice immunized with Can f 1 with Alum were significantly lower than those immunized without Alum. Increases in IFNγ levels after stimulation with Can f 1 or OVA were not remarkable. No influence of genetic backgrounds of BALB/c and C57BL/6 mice was found. Although Can f 1 induced Th2 type immune responses as was also the case for immunization with OVA, an inhibitory effect of Alum on induction of IL13 was observed only in mice immunized with Can f 1. These results suggest that the immune mechanism for allergic sensitization with Can f 1 is different from that with OVA.
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http://dx.doi.org/10.2220/biomedres.40.9DOI Listing
June 2019

Unusual lymph node metastasis from cancer of the thoracic esophagus.

J Surg Case Rep 2018 Aug 14;2018(8):rjy214. Epub 2018 Aug 14.

Department of Digestive and General Surgery, Uonuma Institute of Community Medicine, Niigata University Medical and Dental Hospital, Uonuma Kikan Hospital, Minami-Uonuma, Japan.

A 76-year-old male received concurrent chemoradiotherapy, at a dose of 60 Gy with low-dose 5-fluorouracil, for cT1bN0M0 squamous cell carcinoma of the mid-thoracic esophagus. Because his primary tumor relapsed with mediastinal and right supraclavicular node metastasis 4 months after completion of chemoradiotherapy, right transthoracic esophagectomy with mediastinal and right cervical lymphadenectomy was performed. However, metastatic tumors developed deep beneath the anterior border of the trapezius muscle 2 months after esophagectomy. dissection of the adipose tissue including the tumor and the transverse cervical artery was performed, followed by adjuvant radiotherapy of 50.4 Gy to the area of dissection. The patient died of pneumonia 11 months after metastasectomy, with locally recurrent disease. We have had three cases of this unusual lymph nodes metastasis from cancer of the thoracic esophagus to date and here present the characteristic imaging findings and the possible mechanism of this unusual lymph node metastasis.
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http://dx.doi.org/10.1093/jscr/rjy214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101636PMC
August 2018

Dynamics of sister chromatids through the cell cycle: Together and apart.

J Cell Biol 2018 06 15;217(6):1887-1889. Epub 2018 May 15.

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan

When and how sister chromatid resolution occurs after DNA replication is a fundamental question. Stanyte et al. (2018. https://doi.org/10.1083/jcb.201801157) used CRISPR/Cas9 technology to label and track genomic loci in live cells throughout the cell cycle, shedding light on how replication is linked to mitotic sister chromatid organization.
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http://dx.doi.org/10.1083/jcb.201804091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987729PMC
June 2018

Mice deficient in aldo-keto reductase 1a (Akr1a) are resistant to thioacetamide-induced liver injury.

Toxicol Lett 2018 Sep 12;294:37-43. Epub 2018 May 12.

Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Yamagata, Japan.

Aldehyde reductase (Akr1a) has been reported to be involved in detoxification of reactive aldehydes as well as in the synthesis of bioactive compounds such as ascorbic acid (AsA). Because Akr1a is expressed at high levels in the liver and is involved in xenobiotic metabolism, our objective was to investigate the hepato-protective role of Akr1a in a thioacetamide (TAA)-induced hepatotoxicity model using Akr1a-deficient (Akr1a) mice. Wild-type (WT) and Akr1a mice were injected intraperitoneally with TAA and the extent of liver injury in the acute phase was assessed. Intriguingly, the extent of TAA-induced liver damage was less in the Akr1a mice than in the WT mice. Biomarkers for the ER stress-induced apoptosis pathway were markedly decreased in the livers of Akr1a mice, whereas AsA levels in plasma did not change significantly in any of the mice. In the liver, TAA is converted to reactive metabolites such as TAA S-oxide and then to TAA S, S-dioxide via the action of CYP2E1. In Akr1a mice, CYP2E1 activity was relatively lower than WT mice at the basal level, leading to reactive TAA metabolites being produced at lower levels after the TAA treatment. The levels of liver proteins that were modified with these metabolites were also lower in the Akr1a mice than the WT mice after the TAA treatment. Furthermore, after a lethal dose of a TAA challenge, the WT mice all died within 36 h, whereas almost all of the Akr1a mice survived. These collective results suggest that Akr1a mice are resistant to TAA-induced liver injury, and it follows that the absence of Akr1a might modulate TAA bioactivation.
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http://dx.doi.org/10.1016/j.toxlet.2018.05.015DOI Listing
September 2018

Impaired diversity of the lung microbiome predicts progression of idiopathic pulmonary fibrosis.

Respir Res 2018 02 27;19(1):34. Epub 2018 Feb 27.

Department of Respiratory Medicine and Allergology, Sapporo Medical University School of Medicine, S1W16 Chuoku, Sapporo, 060-8543, Japan.

Background: Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. Although IPF has not been thought to be associated with bacterial communities, recent papers reported the possible role of microbiome composition in IPF. The roles of microbiomes in respiratory functions and as clinical biomarkers for IPF remain unknown. In this study, we aim to identify the relationship between the microbial environment in the lung and clinical findings.

Methods: Thirty-four subjects diagnosed with IPF were included in this analysis. The 16S rDNA was purified from bronchoalveolar lavage fluid obtained at the time of diagnosis and analyzed using next-generation sequencing techniques to characterize the bacterial communities. Furthermore, microbiomes from mice with bleomycin-induced lung fibrosis were analyzed.

Results: The most prevalent lung phyla were Firmicutes, Proteobacteria and Bacteroidetes. Decreased microbial diversity was found in patients with low forced vital capacity (FVC) and early mortality. Additionally, the diversity and relative abundance of Firmicutes, Streptococcaceae, and Veillonellaceae were significantly associated with FVC, 6-min walk distance, and serum surfactant protein D. Bleomycin-induced lung fibrosis resulted in decrease of diversity and alteration of microbiota in PCoA analysis. These results support the observations in human specimens.

Conclusions: This study identified relationships between specific taxa in BALF and clinical findings, which were also supported by experiments in a mouse model. Our data suggest the possibility that loss of microbial diversity is associated with disease activities of IPF.
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http://dx.doi.org/10.1186/s12931-018-0736-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389110PMC
February 2018

Cardiac tamponade communicating with a posterior mediastinal chylocele after esophagectomy.

J Surg Case Rep 2017 Oct 27;2017(10):rjx216. Epub 2017 Oct 27.

Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

A 75-year-old male received neoadjuvant chemotherapy for locally advanced squamous cell carcinoma of the mid-thoracic esophagus, followed by right transthoracic esophagectomy with extended mediastinal lymphadenectomy. Cardiac tamponade developed on postoperative Days 1 and 13, for which emergency ultrasound-guided drainage was required. Pericardial drainage fluid became chylous after administration of polymeric formula. A computed tomography scan demonstrated the presence of a retrocardiac fluid collection, encompassed by the left pulmonary vein and left atrium, descending aorta and vertebral column. Based on these findings, the diagnosis of chylopericardial tamponade communicating with a posterior mediastinal chylocele was made. The ligation of the thoracic duct was successfully performed via the left-sided thoracoscopic approach on postoperative Day 20 and the clinical course after the second operation was uneventful. The possible mechanisms of this exceptionally rare complication after esophagectomy were discussed.
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http://dx.doi.org/10.1093/jscr/rjx216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798138PMC
October 2017

Surfactant protein A down-regulates epidermal growth factor receptor by mechanisms different from those of surfactant protein D.

J Biol Chem 2017 11 27;292(45):18565-18576. Epub 2017 Sep 27.

From the Departments of Biochemistry.

We recently reported that the lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain (CRD) of SP-D and -glycans of EGFR. Here, we report that surfactant protein A (SP-A) also suppresses EGF signaling in A549 human lung adenocarcinoma cells and in CHOK1 cells stably expressing human EGFR and that SP-A inhibits the proliferation and motility of the A549 cells. Results with I-EGF indicated that SP-A interferes with EGF binding to EGFR, and a ligand blot analysis suggested that SP-A binds EGFR in A549 cells. We also found that SP-A directly binds the recombinant extracellular domain of EGFR (soluble EGFR or sEGFR), and this binding, unlike that of SP-D, was not blocked by EDTA, excess mannose, or peptide:-glycosidase F treatment. We prepared a collagenase-resistant fragment (CRF) of SP-A, consisting of CRD plus the neck domain of SP-A, and observed that CRF directly binds sEGFR but does not suppress EGF-induced phosphorylation of EGFR in or proliferation of A549 cells. These results indicated that SP-A binds EGFR and down-regulates EGF signaling by inhibiting ligand binding to EGFR as well as SP-D. However, unlike for SP-D, SP-A lectin activity and EGFR -glycans were not involved in the interaction between SP-A and EGFR. Furthermore, our results suggested that oligomerization of SP-A is necessary to suppress the effects of SP-A on EGF signaling.
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http://dx.doi.org/10.1074/jbc.M117.800771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682966PMC
November 2017

Disruption of the structural and functional features of surfactant protein A by acrolein in cigarette smoke.

Sci Rep 2017 08 16;7(1):8304. Epub 2017 Aug 16.

Department of Biochemistry, Sapporo Medical University, School of Medicine, Hokkaido, 060-8556, Japan.

The extent to which defective innate immune responses contribute to chronic obstructive pulmonary disease (COPD) is not fully understood. Pulmonary surfactant protein A (SP-A) plays an important role in regulating innate immunity in the lungs. In this study, we hypothesised that cigarette smoke (CS) and its component acrolein might influence pulmonary innate immunity by affecting the function of SP-A. Indeed, acrolein-modified SP-A was detected in the lungs of mice exposed to CS for 1 week. To further confirm this finding, recombinant human SP-A (hSP-A) was incubated with CS extract (CSE) or acrolein and then analysed by western blotting and nanoscale liquid chromatography-matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry. These analyses revealed that CSE and acrolein induced hSP-A oligomerisation and that acrolein induced the modification of six residues in hSP-A: His39, His116, Cys155, Lys180, Lys221, and Cys224. These modifications had significant effects on the innate immune functions of hSP-A. CSE- or acrolein-induced modification of hSP-A significantly decreased hSP-A's ability to inhibit bacterial growth and to enhance macrophage phagocytosis. These findings suggest that CS-induced structural and functional defects in SP-A contribute to the dysfunctional innate immune responses observed in the lung during cigarette smoking.
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http://dx.doi.org/10.1038/s41598-017-08588-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559459PMC
August 2017

Surfactant Protein A Inhibits Growth and Adherence of Uropathogenic To Protect the Bladder from Infection.

J Immunol 2017 04 22;198(7):2898-2905. Epub 2017 Feb 22.

Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan;

Surfactant protein A (SP-A) is a multifunctional host defense collectin that was first identified as a component of pulmonary surfactant. Although SP-A is also expressed in various tissues, including the urinary tract, its innate immune functions in nonpulmonary tissues are poorly understood. In this study, we demonstrated that adherence of uropathogenic (UPEC) to the bladder was enhanced in SP-A-deficient mice, which suggests that SP-A plays an important role in innate immunity against UPEC. To understand the innate immune functions of SP-A in detail, we performed in vitro experiments. SP-A directly bound to UPEC in a Ca-dependent manner, but it did not agglutinate UPEC. Our results suggest that a bouquet-like arrangement seems unsuitable to agglutinate UPEC. Meanwhile, SP-A inhibited growth of UPEC in human urine. Furthermore, the binding of SP-A to UPEC decreased the adherence of bacteria to urothelial cells. These results indicate that direct action of SP-A on UPEC is important in host defense against UPEC. Additionally, adhesion of UPEC to urothelial cells was decreased when the cells were preincubated with SP-A. Adhesion of UPEC to urothelial cells is achieved via interaction between FimH, an adhesin located at bacterial pili, and uroplakin Ia, a glycoprotein expressed on the urothelium. SP-A directly bound to uroplakin Ia and competed with FimH for uroplakin Ia binding. These results lead us to conclude that SP-A plays important roles in host defense against UPEC.
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http://dx.doi.org/10.4049/jimmunol.1502626DOI Listing
April 2017

Surfactant protein A (SP-A) and SP-A-derived peptide attenuate chemotaxis of mast cells induced by human β-defensin 3.

Biochem Biophys Res Commun 2017 03 8;485(1):107-112. Epub 2017 Feb 8.

Department of Biochemistry, Sapporo Medical University School of Medicine, S-1 W-17, Chuo-ku, Sapporo, Japan; Department of Chemistry, Sapporo Medical University Center for Medical Education, S-1 W-17, Chuo-ku, Sapporo, Japan. Electronic address:

Human β-defensin 3 (hBD3) is known to be involved in mast cell activation. However, molecular mechanisms underlying the regulation of hBD3-induced mast cell activation have been poorly understood. We previously reported that SP-A and SP-A-derived peptide 01 (SAP01) regulate the function of hBD3. In this study, we focused on the effects of SP-A and SAP01 on the activation of mast cells induced by hBD3. SAP01 directly bound to hBD3. Mast cell-mediated vascular permeability and edema in hBD3 administered rat ears were decreased when injected with SP-A or SAP01. Compatible with the results in rat ear model, both SP-A and SAP01 inhibited hBD3-induced chemotaxis of mast cells in vitro. Direct interaction between SP-A or SAP01 and hBD3 seemed to be responsible for the inhibitory effects on chemotaxis. Furthermore, SAP01 attenuated hBD3-induced accumulation of mast cells and eosinophils in tracheas of the OVA-sensitized inflammatory model. SP-A might contribute to the regulation of inflammatory responses mediated by mast cells during infection.
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http://dx.doi.org/10.1016/j.bbrc.2017.02.028DOI Listing
March 2017

Condensin I-mediated mitotic chromosome assembly requires association with chromokinesin KIF4A.

Genes Dev 2016 Sep 15;30(17):1931-6. Epub 2016 Sep 15.

Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Tokyo 135-8550, Japan;

The chromokinesin KIF4A has been implicated in shaping mitotic chromosomes, but its functional relationship to condensin complexes remains controversial. Here, we found that, in mitosis, KIF4A associates with condensin I but not with condensin II. Mutational analyses indicated that the enrichment of condensin I to chromosomal axes depends on its association with KIF4A in a way that likely involves its motor activity. Remarkably, this interaction is required for condensin I to confer physiological properties to chromosomes. These observations provide an insight into how condensin I is enriched at chromosomal axes and underscore the significance of axial structure in organizing mitotic chromosomes.
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http://dx.doi.org/10.1101/gad.282855.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066236PMC
September 2016

N-glycans of growth factor receptors: their role in receptor function and disease implications.

Clin Sci (Lond) 2016 10;130(20):1781-92

Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, Global Research Cluster, RIKEN, Wako 351-0198, Japan

Numerous signal-transduction-related molecules are secreted proteins or membrane proteins, and the mechanism by which these molecules are regulated by glycan chains is a very important issue for developing an understanding of the cellular events that transpire. This review covers the functional regulation of epidermal growth factor receptor (EGFR), ErbB3 and the transforming growth factor β (TGF-β) receptor by N-glycans. This review shows that the N-glycans play important roles in regulating protein conformation and interactions with carbohydrate recognition molecules. These results point to the possibility of a novel strategy for controlling cell signalling and developing novel glycan-based therapeutics.
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http://dx.doi.org/10.1042/CS20160273DOI Listing
October 2016

Glycation vs. glycosylation: a tale of two different chemistries and biology in Alzheimer's disease.

Glycoconj J 2016 08 21;33(4):487-97. Epub 2016 Jun 21.

Department of Cellular and Molecular Neuropathology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyou-ku, Tokyo, 113-8421, Japan.

In our previous studies, we reported that the activity of an anti-oxidant enzyme, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) became decreased as the result of glycation in vitro and in vivo. Glycated Cu,Zn-SOD produces hydroxyl radicals in the presence of transition metals due to the formation of a Schiff base adduct and a subsequent Amadori product. This results in the site-specific cleavage of the molecule, followed by random fragmentation. The glycation of other anti-oxidant enzymes such as glutathione peroxidase and thioredoxin reductase results in a loss or decrease in enzyme activity under pathological conditions, resulting in oxidative stress. The inactivation of anti-oxidant enzymes induces oxidative stress in aging, diabetes and neurodegenerative disorders. It is well known that the levels of Amadori products and N(e)-(carboxylmethyl)lysine (CML) and other carbonyl compounds are increased in diabetes, a situation that will be discussed by the other authors in this special issue. We and others, reported that the glycation products accumulate in the brains of patients with Alzheimer's disease (AD) patients as well as in cerebrospinal fluid (CSF), suggesting that glycation plays a pivotal role in the development of AD. We also showed that enzymatic glycosylation is implicated in the pathogenesis of AD and that oxidative stress is also important in this process. Specific types of glycosylation reactions were found to be up- or downregulated in AD patients, and key AD-related molecules including the amyloid-precursor protein (APP), tau, and APP-cleaving enzymes were shown to be functionally modified as the result of glycosylation. These results suggest that glycation as well as glycosylation are involved in oxidative stress that is associated with aging, diabetes and neurodegenerative diseases such as AD.
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http://dx.doi.org/10.1007/s10719-016-9690-2DOI Listing
August 2016

Disease-associated glycans on cell surface proteins.

Mol Aspects Med 2016 10 27;51:56-70. Epub 2016 Apr 27.

Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, Global Research Cluster, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan. Electronic address:

Most of membrane molecules including cell surface receptors and secreted proteins including ligands are glycoproteins and glycolipids. Therefore, identifying the functional significance of glycans is crucial for developing an understanding of cell signaling and subsequent physiological and pathological cellular events. In particular, the function of N-glycans associated with cell surface receptors has been extensively studied since they are directly involved in controlling cellular functions. In this review, we focus on the roles of glycosyltransferases that are involved in the modification of N-glycans and their target proteins such as epidermal growth factor receptor (EGFR), ErbB3, transforming growth factor β (TGF-β) receptor, T-cell receptors (TCR), β-site APP cleaving enzyme (BACE1), glucose transporter 2 (GLUT2), E-cadherin, and α5β1 integrin in relation to diseases and epithelial-mesenchymal transition (EMT) process. Above of those proteins are subjected to being modified by several glycosyltransferases such as N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase IV (GnT-IV), N-acetylglucosaminyltransferase V (GnT-V), α2,6 sialyltransferase 1 (ST6GAL1), and α1,6 fucosyltransferase (Fut8), which are typical N-glycan branching enzymes and play pivotal roles in regulating the function of cell surface receptors in pathological cell signaling.
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http://dx.doi.org/10.1016/j.mam.2016.04.008DOI Listing
October 2016

Phosphoproteomic analysis of human mitotic chromosomes identified a chromokinesin KIF4A.

Biomed Res 2016 ;37(2):161-5

Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research (JFCR).

Protein phosphorylation is the prime post-translational modification to drive cell division. Identification of phosphorylated proteins and their related kinases has uncovered molecular networks underlying mitotic processes, including chromosome assembly. Here we aimed to identify phosphoproteins from a mitotic chromosome-enriched lysate biochemically using mass spectrometry. We employed the Polo-box domain (PBD) of Polo-like kinase to tether phosphorylated proteins in the lysate. Resulting candidates included a number of chromosomal proteins that would not be identified unless using chromosome-enriched fractions. Among them, we focused to a chromokinesin KIF4A which becomes concentrated along the longitudinal axis of mitotic condensed chromatids. We found that KIF4A is phosphorylated specifically during mitosis, depending on the activity of Cdk1 and Aurora B, which turned out to be required for KIF4A to interact with condensin I. The molecular link between KIF4A and condensin raises an interesting possibility that the interaction of two distantly related ATPases is triggered by mitotic phosphorylation.
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http://dx.doi.org/10.2220/biomedres.37.161DOI Listing
January 2017

Proteomic and glycomic analyses of a lung-specific protein surfactant protein-D.

Data Brief 2015 Dec 30;5:707-11. Epub 2015 Sep 30.

Diease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

In order to verify the protein enriched from pooled human sera to be a lung-specific protein surfactant protein-D (SP-D), we performed peptide mass fingerprinting (PMF)-based protein identification. MASCOT search results of the obtained PMF unequivocally demonstrated that it is identical to human SP-D. Meanwhile, we performed MALDI-QIT-TOF mass spectrometry-based N-glycomic analysis of the recombinant human SP-D produced in murine myeloma cells. The obtained mass spectra of N-glycans from the recombinant SP-D demonstrated that the recombinant protein is almost exclusively modified with core-fucosylated N-glycans [1].
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http://dx.doi.org/10.1016/j.dib.2015.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773361PMC
December 2015

Fucosylated surfactant protein-D is a biomarker candidate for the development of chronic obstructive pulmonary disease.

J Proteomics 2015 Sep 21;127(Pt B):386-94. Epub 2015 Jul 21.

Disease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Electronic address:

Unlabelled: We previously reported that knockout mice for α1,6-fucosyltransferase (Fut8), which catalyzes the biosynthesis of core-fucose in N-glycans, develop emphysema and that Fut8 heterozygous knockout mice are more sensitive to cigarette smoke-induced emphysema than wild-type mice. Moreover, a lower FUT8 activity was found to be associated with a faster decline in lung function among chronic obstructive pulmonary disease (COPD) patients. These results led us to hypothesize that core-fucosylation levels in a glycoprotein could be used as a biomarker for COPD. We focused on a lung-specific glycoprotein, surfactant protein D (SP-D), which plays a role in immune responses and is present in the distal airways, alveoli, and blood circulation. The results of a glycomic analysis reported herein demonstrate the presence of a core-fucose in an N-glycan on enriched SP-D from pooled human sera. We developed an antibody-lectin enzyme immunoassay (EIA) for assessing fucosylation (core-fucose and α1,3/4 fucose) in COPD patients. The results indicate that fucosylation levels in serum SP-D are significantly higher in COPD patients than in non-COPD smokers. The severity of emphysema was positively associated with fucosylation levels in serum SP-D in smokers. Our findings suggest that increased fucosylation levels in serum SP-D are associated with the development of COPD.

Biological Significance: It has been proposed that serum SP-D concentrations are predictive of COPD pathogenesis, but distinguishing between COPD patients and healthy individuals to establish a clear cut-off value is difficult because smoking status highly affects circulating SP-D levels. Herein, we focused on N-glycosylation in SP-D and examined whether or not N-glycosylation patterns in SP-D are associated with the pathogenesis of COPD. We performed an N-glycomic analysis of human serum SP-D and the results show that a core-fucose is present in its N-glycan. We also found that the N-glycosylation in serum SP-D was indeed altered in COPD, that is, fucosylation levels including core-fucosylation are significantly increased in COPD patients compared with non-COPD smokers. The severity of emphysema was positively associated with fucosylation levels in serum SP-D in smokers. Our findings shed new light on the discovery and/or development of a useful biomarker based on glycosylation changes for diagnosing COPD. This article is part of a Special Issue entitled: HUPO 2014.
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http://dx.doi.org/10.1016/j.jprot.2015.07.011DOI Listing
September 2015

The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway.

Biochem Biophys Res Commun 2014 11 24;454(3):364-8. Epub 2014 Oct 24.

Department of Biochemistry, Sapporo Medical University School of Medicine, Japan.

It has been well documented that activation of the ErbB3-PI3K-Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K-Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.
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http://dx.doi.org/10.1016/j.bbrc.2014.10.086DOI Listing
November 2014

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals.

Biochem Biophys Res Commun 2014 Sep 21;452(1):136-41. Epub 2014 Aug 21.

Department of Biochemistry and Molecular Biology, Yamagata University School of Medicine, Yamagata, Japan.

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.
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http://dx.doi.org/10.1016/j.bbrc.2014.08.072DOI Listing
September 2014

The effects of exercise training on obesity-induced dysregulated expression of adipokines in white adipose tissue.

Int J Endocrinol 2013 4;2013:801743. Epub 2013 Dec 4.

Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.

Obesity is recognized as a risk factor for lifestyle-related diseases such as type 2 diabetes and cardiovascular disease. White adipose tissue (WAT) is not only a static storage site for energy; it is also a dynamic tissue that is actively involved in metabolic reactions and produces humoral factors, such as leptin and adiponectin, which are collectively referred to as adipokines. Additionally, because there is much evidence that obesity-induced inflammatory changes in WAT, which is caused by dysregulated expression of inflammation-related adipokines involving tumor necrosis factor- α and monocyte chemoattractant protein 1, contribute to the development of insulin resistance, WAT has attracted special attention as an organ that causes diabetes and other lifestyle-related diseases. Exercise training (TR) not only leads to a decrease in WAT mass but also attenuates obesity-induced dysregulated expression of the inflammation-related adipokines in WAT. Therefore, TR is widely used as a tool for preventing and improving lifestyle-related diseases. This review outlines the impact of TR on the expression and secretory response of adipokines in WAT.
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http://dx.doi.org/10.1155/2013/801743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867917PMC
June 2014

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice.

Life Sci 2014 Jan 17;95(1):1-8. Epub 2013 Dec 17.

Department of Biochemistry and Molecular Biology, Yamagata University School of Medicine, Yamagata, Japan. Electronic address:

Aims: Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a(-/-)) mice and human Akrla-transgenic (Akr1a(tg/+)) mice experience different anesthetic actions from pentobarbital-prolonged in Akr1a-knockout (Akr1a(-/-)) mice and shortened in human Akrla-transgenic (Akr1a(tg/+)) mice.

Main Methods: We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice.

Key Findings: Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a(-/-) mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a(-/-) mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.

Significance: Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.
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http://dx.doi.org/10.1016/j.lfs.2013.12.004DOI Listing
January 2014