Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3′–5′ exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.     

Real‐time functional optical‐resolution photoacoustic microscopy using high‐speed alternating illumination at 532 and 1064 nm

Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps.      

Changes of butterfly communities after forest fire

Fires change the diversity and composition of insects in forest ecosystems. In the present study, we examined the change of butterfly communities after a fire including the increase of butterfly richness, grassland species, and generalist species, and more changed communities. Butterflies were surveyed for 5 years after the big Uljin fire in 2007. During each year, butterflies were counted monthly by the line transect method from April to October at two sites (burned vs. unburned, ~ 1.5 km routes). Specialist grassland species decreased in the year of the fire but generalist species did not increase significantly. Butterfly richness did not change but butterfly diversity decreased due to a sudden increase of a species, Polygonia c-aureum. The butterfly community in the year of the fire was different from those in later years, showing temporary change of community in the year of the fire. Species composition was significantly different between burned and unburned sites, but this phenomenon cannot be interpreted as an influence of fire due to highly variable species composition of local butterfly assemblages and the non-repetitive sampling site of the present study.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Potent inhibitors of lipoprotein-associated phospholipase A(2): benzaldehyde O-heterocycle-4-carbonyloxime

A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromolar potency for inhibiting Lp-PLA(2) in whole human plasma and isolated human LDL. Based on these results, structure-activity relationship was studied on modification of three parts of R(1), R(2), and R(3) to identify a potent pharmacophore for Lp-PLA(2). In an attempt to introduce various functional groups at R(2) and R(3), we discovered that replacement of less lipophilic groups led to an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R(2) and R(3) had the highest potency with an IC(50) value of 0.05 microM in whole human plasma.     

Lipase-catalyzed synthesis of sorbitol-fatty acid esters at extremely high substrate concentrations

Lipase-catalyzed synthesis of sorbitol-fatty acid esters was performed in eutectic media with extremely high substrate concentrations. Homogeneous eutectic melts of sorbitol and fatty acids of C6-C16 were prepared using an adjuvant mixture. Enhanced homogeneity of mixtures was confirmed by X-ray diffraction analysis. The substrate concentration was 3.63-6.67 M in the eutectic media, whereas in organic media the concentration was below 0.10 M. Esters were synthesized with an immobilized Candida antarctica lipase, and optimum conditions were analyzed. Compared to reactions in organic media, the initial reaction rate of ester synthesis and the overall productivity were significantly enhanced in eutectic media while the conversion yields were similar. Based on the kinetic analysis, highly viscous eutectic media were shown to influence the initial reaction rate and the apparent activation energy resulting in diffusion limitations.     

The forces behind the words: development of the kinetic pen

This paper describes the creation of a Kinetic Pen capable of measuring the six-component force and torque that each of four individual contacts applies to the pen during writing. This was done by staggering the mounting of the four sensors along the long axis of the pen and having an extended arm run from the sensor to the grip site, preventing a clustering of the sensors where the digit tips meet while grasping. The implications of this tool allow handwriting studies to be expanded from two-dimensional pen-tip kinematics to three-dimensional dynamics at each contact point between the hand and pen.     

First finding of a quarantine pest,Atherigona (Acritochaeta) orientalis Schiner (Diptera: Muscidae), in Korea

The invasive quarantine pest fly, Atherigona (Acritochaeta) orientalis Schiner, is observed for the first time in tomato greenhouses in Gyeongsangbuk‐do, Korea. The genus Atherigona Rondani is also newly added to Korean fauna. Allium tuberosum is listed as a new host crop for this species. Some morphological characteristics for accurate identification and host lists are given to provide plant quarantine information for pest management.