Direct Observation and Analysis of TET-mediated Oxidation Processes in a DNA Origami Nanochip

DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as beta-lactams.ArticleJOURNAL OF MEDICAL MICROBIOLOGY. 62:1883-1890 (2013)journal articl

SYNTHESIS OF NIGRICANIN VIA INTRAMOLECULAR BIARYL COUPLING REACTION OF FUNCTIONALIZED PHENYL BENZOATE

A tetracyclic natural product, nigricanin (1), was synthesized through an intramolecular biaryl coupling reaction of the phenyl benzoate derivative which was derived from the corresponding phenol and benzoic acid

Effect of boron deficiency on tip burn and malformed fruit incidence in strawberries

Boron (B) is an essential micro element for plants and plays important roles in the synthesis and functions of cell wall. B deficiency has been reported as one of the causes of fruit malformation in strawberries. We investigated the effect of B deficiency on flower and fruit development of forced strawberries for two cropping seasons (2015-2017). In the second season, B was resupplied for B-deficient plants and we investigated changes in fruit development. When B-free nutrient solutions were supplied, tip burn began to occur in newly emerging leaves and calyx 2 to 3 months later, and fruit malformation including seedy or only partly developed fruits with undeveloped achenes occurred frequently. However, these deficient symptoms were quickly disappeared by supplying B containing nutrient solutions. In conclusion, B nutrition is closely related to the occurrence of fruit malformation through fertility of pollen and pistils, and also development of receptacle tissue in strawberries. It should be possible to reduce fruit malformation in strawberries by proper control of B nutrition.　B（ホウ素）は植物にとって必須な微量要素であり，イチゴの受精不良果発生要因の1 つである．そこで2015年度と2016年度の2 回にわたりB 欠乏がイチゴの受精不良果発生に及ぼす影響について調査した．また，2016年度はB 欠乏処理後にB 回復処理を行い，その後のイチゴの果実形態の変化についても調査した．その結果，B欠乏処理を行うと蒸散機能の低い新葉や花芽においてチップバーンが発生し，種浮き果や部分不受精を主とした受精不良果が多発した．しかし，B 回復処理を行うことでこれらの症状が改善することが明らかになった．B は受精不良果発生に関係しており，欠乏条件下で根から吸収させると急速に若い成長中の組織に転流することが示されたことから，B 栄養をコントロールすることでB 不足によるイチゴの受精不良果発生を軽減できる可能性があると考えられる

Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

<p>Abstract</p> <p>Background</p> <p>We have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from <it>Bifidobacterium adolescentis </it>G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of <it>B. longum </it>NCC2705 and <it>B. adolescentis </it>ATCC 15703 were determined, and <it>cscA </it>gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of <it>cscA </it>gene encoding putative β-FFase from <it>B. adolescentis </it>G1, its expression in <it>E. coli </it>and properties of the recombinant protein are described.</p> <p>Results</p> <p>Using the information of <it>cscA </it>gene from <it>Bifidobacterium adolescentis </it>ATCC 15703, <it>cscA </it>gene from <it>B. adolescentis </it>G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from <it>B. adolescentis </it>G1 was identical to the deduced amino acid sequences of <it>cscA </it>gene from <it>B. adolescentis </it>G1. To confirm the translated product of the <it>cscA </it>gene, the recombinant protein was expressed in <it>Escherichia coli</it>. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The <it>K</it>_m(mM), <it>V</it>_max(μmol/mg of protein/min), <it>k</it>₀(sec^-1) and <it>k</it>₀/<it>K</it>_m(mM^-1sec^-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO₃, SDS, and HgCl₂.</p> <p>Conclusion</p> <p>The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.</p

Effect of boron deficiency on tip burn and malformed fruit incidence in strawberries

Boron (B) is an essential micro element for plants and plays important roles in the synthesis and functions of cell wall. B deficiency has been reported as one of the causes of fruit malformation in strawberries. We investigated the effect of B deficiency on flower and fruit development of forced strawberries for two cropping seasons (2015-2017). In the second season, B was resupplied for B-deficient plants and we investigated changes in fruit development. When B-free nutrient solutions were supplied, tip burn began to occur in newly emerging leaves and calyx 2 to 3 months later, and fruit malformation including seedy or only partly developed fruits with undeveloped achenes occurred frequently. However, these deficient symptoms were quickly disappeared by supplying B containing nutrient solutions. In conclusion, B nutrition is closely related to the occurrence of fruit malformation through fertility of pollen and pistils, and also development of receptacle tissue in strawberries. It should be possible to reduce fruit malformation in strawberries by proper control of B nutrition.　B（ホウ素）は植物にとって必須な微量要素であり，イチゴの受精不良果発生要因の1 つである．そこで2015年度と2016年度の2 回にわたりB 欠乏がイチゴの受精不良果発生に及ぼす影響について調査した．また，2016年度はB 欠乏処理後にB 回復処理を行い，その後のイチゴの果実形態の変化についても調査した．その結果，B欠乏処理を行うと蒸散機能の低い新葉や花芽においてチップバーンが発生し，種浮き果や部分不受精を主とした受精不良果が多発した．しかし，B 回復処理を行うことでこれらの症状が改善することが明らかになった．B は受精不良果発生に関係しており，欠乏条件下で根から吸収させると急速に若い成長中の組織に転流することが示されたことから，B 栄養をコントロールすることでB 不足によるイチゴの受精不良果発生を軽減できる可能性があると考えられる

Comparison of INTREPID® balanced and hybrid tips on anterior capsule rupture in ex vivo porcine eyes.

Phacoemulsification has emerged as the global standard for cataract surgery, and various novel methods, tools, and agents have promoted surgical efficiency and reduced complications. Conventionally, the phaco tip, which cleaves and aspirates the cataractous lens, has been mainly constructed of metal. In this study, the risk of anterior capsule rupture was evaluated under conditions of different power modes, longitudinal (Mode-L), torsional (Mode-T), or both (Mode-LT), and different aspiration powers (0 or 200 mmHg), using a traditional metal phaco tip (Group-M) or a new phaco tip with a high-strength polymer overmold on the needle edge (Group-P), which was developed to reduce the risk of capsule rupture. One hundred twenty porcine eyes were used for experiments within a setting of typical human physiological intraocular pressure. We found that Group-M showed capsule rupture with a smaller ultrasound power than did Group-P, regardless of power mode or aspiration power. In Group-M, there was no significant difference in risk of capsule rupture among power modes, however in Group-P, capsule rupture was least likely to occur with Mode-T. These results provide useful information for inexperienced ophthalmologists to improve surgical safety

Neural Networks Integrated Circuit for Biomimetics MEMS Microrobot

In this paper, we will propose the neural networks integrated circuit (NNIC) which is the driving waveform generator of the 4.0, 2.7, 2.5 mm, width, length, height in size biomimetics microelectromechanical systems (MEMS) microrobot. The microrobot was made from silicon wafer fabricated by micro fabrication technology. The mechanical system of the robot was equipped with small size rotary type actuators, link mechanisms and six legs to realize the ant-like switching behavior. The NNIC generates the driving waveform using synchronization phenomena such as biological neural networks. The driving waveform can operate the actuators of the MEMS microrobot directly. Therefore, the NNIC bare chip realizes the robot control without using any software programs or A/D converters. The microrobot performed forward and backward locomotion, and also changes direction by inputting an external single trigger pulse. The locomotion speed of the microrobot was 26.4 mm/min when the step width was 0.88 mm. The power consumption of the system was 250 mWh when the room temperature was 298 K