Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe: development of a fed-batch fermentation and purification process

A fed-batch fermentation process has been developed to enable the production of large quantities of recombinant human lysosomal acid lipase (hLAL; EC 3.1.1.13), in Schizosaccharomyces pombe, for preclinical studies as a potential enzyme therapy drug. Recombinant S. pombe, clone ASP397-21, expressed enzymatically active hLAL in the secreted form. A feedback fed-batch system was used to determine the optimal feed rate of a 50% glucose solution used as the carbon source. The feed rate of the glucose solution was calculated by a computer-aided system according to the equation; F=q(sf)(VX)/S(in) (q(sf), specific substrate feed rate [gram substrate/gram dry cell weight/h]; V, volume of culture broth [l]; X, cell density [gram dry cell weight/l]; S(in), concentration of growth limiting substrate in feed solution [gram substrate/gram feed solution]). At the time of the initial consumption of glucose in the batch-phase culture, the nutrient supply was automatically initiated by means of monitoring the respiratory quotient change. The obtained profile of the feed rate was applied to the feed forward control fermentation. Finally, the cells were grown up to >50 g dry cell weight/l, and the hLAL expression level was approximately 16,000 U/l. Expressed hLAL protein was purified in a two-step process by hydrophobic interaction and anion exchange chromatographies. Purified recombinant hLAL exhibited a 90-150 kDa broad band upon SDS-PAGE with specific activity of about 300 U/mg. After endoglycosidase H treatment, the band converged to 45 kDa, equal to the calculated molecular weight, suggesting that hLAL produced in S. pombe was hyper-glycosylated. N-terminal analysis of de-glycosylated hLAL revealed that the signal sequence of hLAL was correctly processed in S. pombe.     

Preparation and characterization of carbonaceous materials containing nitrogen as electrochemical capacitor

Carbonaceous materials containing nitrogen (C/N materials) were prepared by a pyrolysis of 2,3,6,7-tetracyano-1,4,5,8-tetraazanaphthalene (CAN). A C/N material prepared by the pyrolysis of CAN at 1070 K (CAN-1070 K) had a C/N atomic ratio of 3.0 and a non-crystalline carbonaceous structure with a BET surface area of 880 m² g⁻¹. The material CAN-1070 K showed large capacitances of 160–180 F g⁻¹ and 110–120 F cm⁻³ in case of current density of 10 mA cm⁻² (2 A g⁻¹) by using three-electrode cell in 1 M H₂SO₄ aqueous solution, in comparison with that of activated carbon (160 F g⁻¹ and 55 F cm⁻³) having BET surface area of 2300 m² g⁻¹. ESCA study indicated that pyridinic and quarternary nitrogen atoms existed in the C/N materials, which could result in producing a pseudo-capacitance in addition to the electric double layer capacitance. Also introduction of nitrogen into the carbonaceous material could enhance the wettability of material, which might also improve the capacitance.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.     

Improvements in time resolution and signal-to-noise ratio in a compact pico-second pulse radiolysis system

A compact pico-second pulse radiolysis system has been developing at Waseda University for studying primary processes in radiation chemistry. The system is composed of a photo-injector system and a pico-second all-solid-state laser system. An infrared (IR) and an ultraviolet (UV) laser pulses are obtained from mode-locked Nd:YLF laser system and used for generation of the white light continuum as a probe light and the irradiation to the Cu cathode of a photo-cathode RF-gun, respectively. To improve signal-to-noise (S/N) ratio and time resolution of this pulse radiolysis system, we optimized both probe light and pump electron beam. As a result, our pico-second pulse radiolysis system has been enough to study the primary processes of radiation chemistry. The experimental results and the improvements of our system are described in this paper.     

An OTFT‐driven rollable OLED display

Abstract— An 80‐μm‐thick rollable AMOLED display driven by an OTFT is reported. The display was developed so as to be rollable in one direction with an integrated OTFT gate driver circuit. It was successfully operated by an originally developed organic semiconductor, a peri‐xanthenoxanthene derivative. The display retained its initial electrical properties and picture quality even after being subjected to 1000 cycles of a roll‐up‐and‐release test with a radius of 4 mm.     

Making 2D Map of Environments Based upon Routes Scenes

This paper proposes a method for making a map of large scale environment based upon route scenes, assuming that the topological relation of routes at intersections is known. A panoramic representation is used for describing route scenes, and the number of routes connecting at an intersection is assumed to be known. The idea is to decompose a 2D graph into a number of closed loops. By detecting the closed loops and storing the relation among them, we can describe the 2D map based upon route scenes. A robot can obtain a closed loop by taking the same turn (leftmost for example) at every intersection when it moves along routes. According to the information on routes at intersections, the robot can select unmoved routes for finding new closed loops. By fusing new closed loops with found ones, the robot can, further, build the map of environments. The effectiveness of our method are shown by experiment in a real-world environment.     

Inhibition of the Phospholipase Cε–c-Jun N-Terminal Kinase Axis Suppresses Glioma Stem Cell Properties

Glioma stem cells (GSCs), the cancer stem cells of glioblastoma multiforme (GBM), contribute to the malignancy of GBM due to their resistance to therapy and tumorigenic potential; therefore, the development of GSC-targeted therapies is urgently needed to improve the poor prognosis of GBM patients. The molecular mechanisms maintaining GSCs need to be elucidated in more detail for the development of GSC-targeted therapy. In comparison with patient-derived GSCs and their differentiated counterparts, we herein demonstrated for the first time that phospholipase C (PLC)ε was highly expressed in GSCs, in contrast to other PLC isoforms. A broad-spectrum PLC inhibitor suppressed the viability of GSCs, but not their stemness. Nevertheless, the knockdown of PLCε suppressed the survival of GSCs and induced cell death. The stem cell capacity of residual viable cells was also suppressed. Moreover, the survival of mice that were transplanted with PLCε knockdown-GSCs was longer than the control group. PLCε maintained the stemness of GSCs via the activation of JNK. The present study demonstrated for the first time that PLCε plays a critical role in maintaining the survival, stemness, and tumor initiation capacity of GSCs. Our study suggested that PLCε is a promising anti-GSC therapeutic target.     

3D reconstruction considering calculation time reduction for linear trajectory shooting and accuracy verification with simulator

Artificial Life and Robotics - Photogrammetry is a three-dimensional (3D) reconstruction from images. In photogrammetry, when each image captures the features of the target object for 3D...     

Effect of meso Positioned Substituents on the Stability and Photodynamic Activity of Lipid-Membrane-Incorporated Porphyrin Derivatives

In this study we prepared aqueous solutions of lipid-membrane incorporated tetraarylporphyrins and tetrapyridylporphyrin (LMIPors) by the injection method using dimethyl sulfoxide. The porphyrins with proton-donor groups at the meso position afforded stable aqueous solutions of LMIPors. However, although tetrakis(carboxyphenyl)porphyrin was scarcely incorporated in lipid membranes, it was soluble in water. Among these LMIPors, the photodynamic activity of tetrakis(hydroxyphenyl)porphyrin was higher than that of tetrakis(aminophenyl)porphyrin. This was attributed to the self-aggregation of a part of tetrakis(aminophenyl)porphyrin in the liposomes, which induced self-quenching and the consequent decrease of its photodynamic activity.     

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.