An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward P_fr → P_o transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse P_o → P_fr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm⁻¹. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.     

Pore Structure and Thermal Stability of Mesoporous Mullite Fibers Prepared by Crystallization and Selective Leaching of Al2O3-SiO2 Glass Fibers

Porous mullite fibers were prepared by crystallization and selective leaching of Al2O3-SiO2 glass fibers using buffered HF-NH4F(BHF) aqueous solutions. The optimum concentration of BHF solution for selective leaching of the fibers was 0.9 mass% HF and 17.0 mass% NH4F. By firing at 1000–1300°C, the glass fibers changed into composite texture of mullite and glassy phase. Since the pores in the fibers were formed by selective leaching of glassy phase among mullite grains, they were tunable by changing the firing conditions of fibers. Pore size of the samples changed from around 4 nm in the 1100°C fired sample to 16 and 40 nm in the 1200 and 1300°C fired samples, respectively. The highest specific surface area obtained was around 30 m2/g, when the fibers were heat treated at 1200°C for 24 h and leached for 20 h in 0.9 mass% HF-17.0 mass% NH4F solution. From the thermal stability tests of the porous mullite fibers, its specific surface area was found to be maintained up to 1200–1300°C.     

Ion beam induced luminescence of polyethylene terephthalate foils under MeV H and He ion bombardment

The evolution of the ion beam induced luminescence (IBIL) of the polyethylene terephthalate (PET) foils was studied under the irradiation of H and He ions of MeV energy. The optical and chemical changes of the samples were also examined by photo-stimulated luminescence and optical absorption measurements after the irradiation. A prominent broad emission peak of IBIL appeared at around 380 nm, and its intensity monotonically decreased during the ion irradiation. The decay curves of the emission intensity were quantitatively explained as a function of the electronic energy deposition of the incident H and He ions. On the contrary, to the decrease of the main emission peak, a growth of new peaks was observed in the wavelengths between 500 and 600 nm.     

Sweep‐out flow of film condensation on vertical plates with horizontal grooves

We have investigated fluid flow characteristics of film‐wise condensation on vertical plates with horizontal periodic grooves. Condensate stays at the edge of the grooves due to the surface tension. The condensate starts to flow, however, when the balance between the surface tension and the condensate's own weight is broken. It is found that the condensate flows downward successively and periodically from the top part of the plate as a group. In addition, we have obtained the relation between the frequencies of the periodic flow and the degree of sub‐cooling for two different pitches of the grooves. © 2009 Wiley Periodicals, Inc. Heat Trans Asian Res; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/htj.20249     

Molecular structures of gellan gum imaged with atomic force microscopy (AFM) in relation to the rheological behavior in aqueous systems in the presence of sodium chloride

Aqueous solutions of gellan gum with comparable molecular mass but with different acyl contents were investigated by atomic force microscopy and rheological measurements in the presence of sodium chloride (NaCl). Results obtained were discussed in relation to our previous report using potassium chloride (KCl) as an added salt. For a low-acyl sample, continuous fibrous network structures were identified microscopically as in the case of KCl. The network structures were more heterogeneous than those formed with KCl in terms of the height distribution of molecular assemblies. Rheological thermal hysteresis between sol–gel transitions was detected as in the case of KCl. The storage modulus (G′) of the gelled system was ca. 15% of the corresponding data with KCl at 20 °C. For a high-acyl sample, no continuous network structures were identified but branches with observable ends were identified as in the case of KCl. The hysteresis was less evident than the corresponding data with KCl and for the low-acyl sample with NaCl. Also, G′ values at 20 °C were ca. 30% and 20% of the corresponding data with KCl and for the low-acyl sample with NaCl, respectively. Continuousness and homogeneity of network structures related to the hysteresis and elasticity of the system, respectively.     

Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.     

Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system

Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.     

A novel spacer‐free panel structure and glass for FEDs

Abstract— A new design for an FED envelope, composed of box‐shaped front glass, sheet‐like rear glass, and metal members, has been devised. This design structure is effective in reducing tensile stress induced by vacuum at the sealing points. Also, a new glass composition, a new physical tempering method, and a lead‐free hermetic sealing material have been developed. As a result, a novel lightweight spacer‐free panel structure for FEDs has been developed by integrating these new technologies. It will assist in maximizing the essential advantages of FED such as high image quality, high reliability, and low cost.     

A histochemical method using a substrate of beta-glucuronidase for detection of genetically modified papaya

A histochemical assay for detecting genetically modified (GM) papaya (derived from Line 55-1) is described. GM papaya, currently undergoing a safety assessment in Japan, was developed using a construct that included a beta-glucuronidase (GUS) reporter gene linked to a virus coat protein (CP) gene. Histochemical assay was used to visualize the blue GUS reaction product from transgenic seed embryos. Twelve embryos per fruit were extracted from the papaya seeds using a surgical knife. The embryos were incubated with the substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc) in a 96-well microtiter plate for 10-15 hours at 37 degrees C. Seventy-five percent of GM papaya embryos should turn blue theoretically. The histochemical assay results were completely consistent with those from a qualitative polymerase chain reaction (PCR) method developed by this laboratory. Furthermore, the method was validated in a five-laboratory study. The method for detection of GM papaya is rapid and simple, and does not require use of specialized equipment.