In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.

In recent years, multidrug resistance of Escherichia coli has become a serious problem. However, resistance to fosfomycin (FOM) has been low. We screened E. coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanisms of resistance and reduced susceptibility of these strains. Ten strains showing reduced FOM susceptibility (MIC ≥ 8 μg/mL) in 211 clinical isolates were found and examined. Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) and mutations in murA that underlie high resistance to FOM were not observed. We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter. In ten strains, nine showed lack of growth on M9 minimum salt agar supplemented with G6P. Eight of the ten strains showed fluctuated induction by G6P of uhpT that encodes G6P transporter expression. Nucleotide sequences of the uhpT, uhpA, glpT, ptsI, and cyaA shared several deletions and amino acid mutations in the nine strains with lack of growth on G6P-supplemented M9 agar. In conclusion, reduction of uhpT function is largely responsible for the reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA. However, there are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear.

Tigecycline (TGC) is a last-line drug for multidrug-resistant Enterobacteriaceae. We investigated the mechanism(s) underlying TGC nonsusceptibility (TGC resistant/intermediate) in Escherichia coli clinical isolates. The MIC of TGC was determined for 277 fluoroquinolone-susceptible isolates (ciprofloxacin [CIP] MIC, Ͻ0.125 mg/liter) and 194 fluoroquinolone-resistant isolates (CIP MIC, Ͼ2 mg/liter). The MIC 50 and MIC 90 for TGC in fluoroquinolone-resistant isolates were 2-fold higher than those in fluoroquinolone-susceptible isolates (MIC 50 , 0.5 mg/liter versus 0.25 mg/liter; MIC 90 , 1 mg/liter versus 0.5 mg/liter, respectively). Two fluoroquinolone-resistant isolates (O25b:H4-ST131-H30R and O125:H37-ST48) were TGC resistant (MICs of 4 and 16 mg/liter, respectively), and four other isolates of O25b:H4-ST131-H30R and an isolate of O1-ST648 showed an intermediate interpretation (MIC, 2 mg/liter). No TGC-resistant/intermediate strains were found among the fluoroquinolone-susceptible isolates. The TGC-resistant/intermediate isolates expressed higher levels of acrA and acrB and had lower intracellular TGC concentrations than susceptible isolates, and they possessed mutations in acrR and/or marR. The MICs of acrAB-deficient mutants were markedly lower (0.25 mg/liter) than those of the parental strain. After continuous stepwise exposure to CIP in vitro, six of eight TGC-susceptible isolates had reduced TGC susceptibility. Two of them acquired TGC resistance (TGC MIC, 4 mg/liter) and exhibited expression of acrA and acrB and mutations in acrR and/or marR. In conclusion, a population of fluoroquinolone-resistant E. coli isolates, including major extraintestinal pathogenic lineages O25b:H4-ST131-H30R and O1-ST648, showed reduced susceptibility to TGC due to overexpression of the efflux pump AcrAB-TolC, leading to decreased intracellular concentrations of the antibiotics that may be associated with the development of fluoroquinolone resistance.

No abstract

SIRT1 is a mammalian ortholog of the yeast enzyme Sir2, which is an NAD +-dependent deacetylase of histones, p53, FOXO, NF-κB, PGC-1α, and other transcription factors. The Sir2 protein is reported as a longevity protein in yeast. Resveratrol, a polyphenol isolated from various types of plant families, particularly the Vitaceae family, is a known naturally occurring SIRT1 activator. In this study, we evaluated the effects of four types of resveratrol dimers and four types of tetramers isolated from vitaceous plants, and one type of resveratrol tetramer isolated from a dipterocarpaceous plant on purified human SIRT1 enzyme activity. Of the resveratrol dimers examined, (+)-ε-viniferin and pallidol exhibited no effect on SIRT1 enzyme activity, whereas (+)-ampelopsin B and (−)-ampelopsin F showed inhibitory activity on SIRT1. However, all the resveratrol tetramers examined, i.e., (+)-vitisin A, (−)-vitisin B, (+)-hopeaphenol, (−)-hopeaphenol, and (−)-isohopeaphenol markedly inhibited the human SIRT1 enzyme activity. (+)-Hopeaphenol exhibited the most potent inhibitory activity, which was comparable with that exhibited by a known SIRT1 inhibitor suramin. Since SIRT1 inhibitors reportedly possess anticancer activity, (+)-hopeaphenol and other resveratrol oligomers can be used as a seed compound for anticancer drugs.

No abstract

Assuming that the 0.6-μm silicon-on-insulator (SOI) complementary metal–oxide–semiconductor (CMOS) technology, different Si-based temperature sensors such as metal-oxide-semiconductor field-effect transistor (MOSFET) (n-channel and p-channel), pn-junction diode (with p-body doping and without doping), and resistors (n+ or p+ single crystalline Si and n+ polycrystalline Si) were designed and characterized for its possible use in 1-THz antenna-coupled bolometers. The use of a half-wave dipole antenna connected to the heater end was assumed, which limited the integrated temperature sensor/heater area to be 15 × 15 µm. Our main focus was to evaluate the performances of the temperature sensor/heater part, and the optical coupling between the incident light and heater via an antenna was not included in the evaluation. The electrothermal feedback (ETF) effect due to the bias current was considered in the performance estimation. A comparative analysis of various SOI bolometers revealed the largest responsivity (Rv) of 5.16 kV/W for the n-channel MOSFET bolometer although the negative ETF in MOSFET reduced the Rv. The noise measurement of the n-channel MOSFET showed the NEP of 245 pW/Hz1/2, which was more than one order of magnitude smaller than that of the n+ polycrystalline Si resistive bolometer (6.59 nW/Hz1/2). The present result suggests that the n-channel MOSFET can be a promising detector for THz applications.