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Kazuhiro Fujiwara - One of the best experts on this subject based on the ideXlab platform.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Naomichi Fujiuchi - One of the best experts on this subject based on the ideXlab platform.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Nobuyuki Matoba - One of the best experts on this subject based on the ideXlab platform.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Ryo Matsuda - One of the best experts on this subject based on the ideXlab platform.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g−1 relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW−1, was comparable to the mean HA yield of 846 μg gFW−1 generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. Biotechnol. Bioeng. 2015;9999: 1–6. © 2015 Wiley Periodicals, Inc.

  • removal of bacterial suspension water occupying the Intercellular Space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a nicotiana benthamiana transient gene expression system
    Biotechnology and Bioengineering, 2016
    Co-Authors: Naomichi Fujiuchi, Nobuyuki Matoba, Ryo Matsuda, Kazuhiro Fujiwara
    Abstract:

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying Intercellular Space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Michele Cicala - One of the best experts on this subject based on the ideXlab platform.

  • dilated Intercellular Space diameter as marker of reflux related mucosal injury in children with chronic cough and gastro oesophageal reflux disease
    Alimentary Pharmacology & Therapeutics, 2014
    Co-Authors: Osvaldo Borrelli, V. Mancini, Mentore Ribolsi, Barbara Bizzarri, Keith J. Lindley, S. Emerenziani, Nikhil Thapar, Gian Luigi Deangelis, Michele Cicala
    Abstract:

    Summary Background The diagnostic corroboration of the relationship between gastro-oesophageal reflux disease (GERD) and chronic cough remains challenging. Aims To compare oesophageal mucosal Intercellular Space diameter (ISD) in children with GERD, children with gastro-oesophageal reflux (GER)-related cough (GrC) and a control group, and to explore the relationship between baseline impedance levels and dilated ISD in children with GER-related cough. Methods Forty children with GERD, 15 children with GrC and 12 controls prospectively underwent oesophagogastroduodenoscopy (EGD) with oesophageal biopsies taken 2–3 cm above squamocolumnar junction. ISD were quantified using transmission electron microscopy. Impedance-pH monitoring with evaluation of baseline impedance in the most distal impedance channel was performed in both patient groups. Results A significant difference in mean ISD values was found between GrC patients (0.9 ± 0.2 μm) and controls (0.5 ± 0.2 μm, P < 0.001), whereas there was no difference between GrC and GERD group (1 ± 0.3 μm, NS). No difference was found in the mean ISD between GrC children with or without pathological oesophageal acid exposure time (1 ± 0.3 vs. 0.9 ± 0.2 μm), and there was no correlation between ISD and any reflux parameter. Finally, there was no correlation between ISD and distal baseline impedance values (r:−0.35; NS). Conclusions In children with reflux-related cough, dilated Intercellular Space diameter appears to be an objective and useful marker of oesophageal mucosal injury regardless of acid exposure, and its evaluation should be considered for those patients where the diagnosis is uncertain. In children with reflux-related cough, baseline impedance levels have no role in identifying reflux-induced oesophageal mucosal ultrastructural changes.

  • Oesophageal mucosal Intercellular Space diameter and reflux pattern in childhood erosive and non-erosive reflux disease
    Digestive and Liver Disease, 2012
    Co-Authors: V. Mancini, Mentore Ribolsi, Massimo Gentile, Gianluigi De’angelis, Barbara Bizzarri, Keith J. Lindley, Salvatore Cucchiara, Michele Cicala, Osvaldo Borrelli
    Abstract:

    Abstract Background and aims We sought to compare Intercellular Space diameter in children with non-erosive and erosive reflux disease, and a control group. We also aimed to characterize the reflux pattern in erosive and non-erosive reflux disease patients, and to explore the relationship between Intercellular Space diameter values and reflux parameters. Methods Twenty-four children with non-erosive reflux disease, 20 with erosive reflux disease, and 10 controls were prospectively studied. All patients and controls underwent upper endoscopy. Biopsies were taken at 2–3 cm above the Z-line, and Intercellular Space diameter was measured using transmission electron microscopy. Non-erosive and erosive reflux disease patients underwent impedance-pH monitoring. Results Mean Intercellular Space diameter values were significantly higher in both non-erosive (0.9 ± 0.2 μm) and erosive reflux disease (1 ± 0.2 μm) compared to controls (0.5 ± 0.2 μm, p Conclusions Dilated Intercellular Space diameter seems to be a useful and objective marker of oesophageal damage in paediatric gastro-oesophageal reflux disease, regardless of acid exposure. In childhood, different gastro-oesophageal reflux disease phenotypes cannot be discriminated on the basis of reflux pattern.

  • Intercellular Space diameters of the oesophageal epithelium in NERD patients: head to head comparison between light and electron microscopy analysis.
    Digestive and Liver Disease, 2008
    Co-Authors: Mentore Ribolsi, Massimo Gentile, Giuseppe Perrone, R. Caviglia, S. Emerenziani, M.p. Luca Guarino, T. Petitti, Michele Cicala
    Abstract:

    Abstract Background Dilation of Intercellular Space diameters of oesophageal epithelium detected at transmission electron microscopy morphometry is a marker of tissue injury in non-erosive reflux disease patients. Semi-quantitative evaluation of Intercellular Space diameters using light microscopy seems to provide promising results. Aim/methods To comparatively evaluate Intercellular Space diameters in the same patients, by means of morphometry and semi-quantitative analysis, both on light microscopy and transmission electron microscopy microphotographs, biopsies were taken in 29 non-erosive reflux disease patients at distal and proximal oesophagus. Twelve asymptomatic controls underwent the same protocol. Results Morphometric analysis on transmission electron microscopy microphotographs showed mean Intercellular Space diameter values of patients, at distal and proximal oesophagus, 3- and 2-fold, respectively, higher than those in controls ( p Conclusions Intercellular Space diameter morphometric analysis at light microscopy is widely available, allows Intercellular Space diameter to be quantitatively measured with good sensitivity and specificity and could represent a useful tool in non-erosive reflux disease diagnosis. Despite satisfactory sensitivity, the semi-quantitative score at light microscopy is hampered by much lower specificity than transmission electron microscopy- and light microscopy-morphometry.

  • Dilated Intercellular Spaces and acid reflux at the distal and proximal oesophagus in patients with non-erosive gastro-oesophageal reflux disease
    Alimentary Pharmacology & Therapeutics, 2007
    Co-Authors: R. Caviglia, Mentore Ribolsi, Massimo Gentile, S. Emerenziani, T. Petitti, Carla Rabitti, Michele Pier Luca Guarino, Michele Cicala
    Abstract:

    Summary Background  Acid exposure of proximal oesophagus and dilated Intercellular Space diameters of oesophageal epithelium are relevant in the perception of gastro-oesophageal reflux. Aim  To explain the relationship between gastro-oesophageal reflux disease symptoms, acid exposure and Intercellular Space diameter along the oesophageal epithelium and to assess time-related variability of Intercellular Space diameter. Methods  Thirty-three non-erosive reflux disease (NERD), six erosive oesophagitis patients and 12 asymptomatic controls underwent oesophageal manometry and 24-h dual-channel oesophageal pH-monitoring following endoscopy. Biopsies were taken 5 cm above the LES and 10 cm below the UES, at comparable levels, as pH sensors. A total of 100 Intercellular Space diameters per patient/control were measured blindly at transmission electron microscopy. In 15 patients, the investigation was repeated after 1 year. Results  In all NERD patients, acid exposure was higher at mid-proximal oesophagus (P