AN EFFICIENT SYSTEM FOR THE ASYMMETRIC ACYLATION OF (R,S)-3-n-BUTYLPHTHALIDE CATALYZED BY NOVOZYME 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran–hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee_S), enantiomeric excess of product (ee_P), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30°C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee_S and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

Granulation of activated sludge in a continuous flow airlift reactor by strong drag force

Most aerobic granule cultivation has been based on the sequencing batch reactor (SBR) and then the factors that affect aerobic granulations were developed in the SBR. However, little work has been done to cultivate aerobic granules in a continuous-flow bioreactor with simple structure that is realistic for engineering. This work is the first to cultivate aerobic granules in a continuous flow airlift fluidized bed reactor (CAFB) possesses a very simple structure and without settling time and starvation time controlling. The configuration of CAFB was the simplest continuous-flow aerobic granular bioreactor reported by now. The majority of granules could be formatted in the CAFB after 12 days cultivation. The effluent COD concentration maintained at 50 ± 10 mg/L for the variable COD loading rate of 3.5 g COD/L/d and 4.8 g COD/L/d, which confirmed that the CAFB performed good anti-shock abilities. CAFB performed good nitrification ability, however, little denitrification was found under the operating conditions of this study. The shear stress acting on the solid phase were hundreds of times stronger in the CAFB than in the SBR at the same aeration strength. It seems CAFB is very efficient for granulation due to the strong shear-force exertion, which is promising for continuous-flow aerobic granular bioreactor. Protein, positive to the hydrophobicity, was predominant in extracellular polymeric substances in the granules, and favored the granules formation in the CAFB combined with the polysaccharides. However, filamentous bulking always happened in 35 days operation of the CAFB, thus further study on the stability of this bioreactor is urgently necessary.     

Secretion of truncated recombinant rabies virus glycoprotein with preserved antigenic properties using a co-expression system in Hansenula polymorpha

Rabies virus infection remains a serious public health threat in the developing world, where cost-concerns make wide-scale public health interventions impractical. The development of novel and inexpensive ELISA diagnostic antigens is critical in early detection and prevention of complications. The transmembrane glycoprotein (G) of rabies virus (RV) contains an external domain capable of inducing the synthesis of anti-rabies, virus-neutralizing antibodies, in infected or immunized hosts. In our study, the external G domain was synthesized and fused in-frame with a polyhistidine-tag coding sequence present in the expression plasmid. Soluble truncated recombinant G was secreted in Hansenula polymorpha (H. polymorpha) using H. polymorpha-derived calnexin (HpCNE1) overproduction and found to be correctly N-glycosylated. The truncated recombinant G was purified from cell culture supernatant by Ni-agarose affinity chromatography and when compared with the full-length glycoprotein, found to be similarly immunogenic in vaccinated rabbits. These results subsequently led us to explore the potential of truncated recombinant G as a diagnostic antigen in ELISA. Our results show that the truncated recombinant G can detect antibodies directed to both whole virion and native glycoprotein. More sophisticated applications of truncated recombinant G would profit from the correctly N-glycosylated and soluble monomer.     

Development of W/O Microemulsion for Transdermal Delivery of Iodide Ions

The objective of this study was to develop a water-in-oil (w/o) microemulsion which can be utilized as a transdermal delivery for iodide ions. Several w/o microemulsion formulations were prepared utilizing Span 20, ethanol, Capryol 90®, and water. The selected formulations had 5%, 10%, 15%, 20%, and a maximum of 23% w/w water content. Potassium iodide (KI) was incorporated in all formulations at 5% w/v. Physicochemical characterizations were conducted to evaluate the structure and stability. These studies included: mean droplet size, pH, viscosity, conductivity, and chemical stability tests. In vitro human skin permeation studies were conducted to evaluate the diffusion of the iodide ion through human skin. The w/o microemulsion formulations were stable and compatible with iodide ions with water content ranging from 5% to 23% w/w. The addition of KI influenced the physicochemical properties of microemulsion as compared to blank microemulsion formulations. In vitro human skin permeation studies indicated that selected formulations improved iodide ion diffusion significantly as compared to control (KI solution; P value < 0.05). Iodide ions were entrapped within the aqueous core of w/o microemulsion. Span 20, ethanol and Capryol 90 protected the iodide ions against oxidation and formed a stable microemulsion. It is worth to note that according to Hofmeister series, iodide ions tend to lower the interfacial tension between water and oil and consequently enhance overall stability. This work illustrates that microemulsion system can be utilized as a vehicle for the transdermal administration of iodide.KEY WORDS: iodide, microemulsion, skin permeation, transdermal     

Hydroxylation of quinocetone and carbadox is mediated by CYP1As in the chicken (Gallus gallus)

Quinoxaline derivatives (quinoxalines) comprise a class of drugs that have been widely used as animal antimicrobial agents and feed additives. Although the metabolism of quinoxaline drugs has been mostly studied using chicken liver microsomes, the biochemical mechanism of biotransformation of these chemicals in the chicken has yet to be characterized. In this study, using bacteria produced enzymes, we demonstrated that both CYP1A4 and CYP1A5 participate in the oxidative metabolism of quinoxalines. For CYP1A5, three hydroxylated metabolites of quinocetone were generated. In addition, CYP1A5 is able to hydroxylate carbadox. For CYP1A4, only one hydroxylated product of quinocetone on the phenyl ring was identified. Neither CYP1A5 nor CYP1A4 showed hydroxylation activity towards mequindox and cyadox. Our results suggest that CYP1A4 and CYP1A5 have different and somewhat overlapping substrate specificity in quinoxaline metabolism, and CYP1A5 represents a crucial enzyme in hydroxylation of both quinocetone and carbadox.     

Fosmid library construction and end sequences analysis of the Pacific oyster,Crassostrea gigas

The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies.     

Improvements in the primary culture of neonate rat myocardial cells by study of the mechanism of endoplasmic reticulum stress

We previously found that endoplasmic reticulum stress (ERS) might be exhibited in the conventional protocol of the primary culture of neonate rat myocardial cells (NRMCs) and that the high glucose concentration (25 mmol/L) in the culture medium might be the cause. Here, we investigated if the high concentration of glucose might influence ERS in myocardial cells during culture. GRP78 expression (ERS marker) was similar in groups with tunicamycin (TM) and without TM in high glucose cultured cells (p?>?0.01). Different glucose concentrations elicited different GRP78 expressions according to analyses of protein and RNA levels, which showed ERS in H/H groups. Finally, we found that GRP78 expression was higher in TM groups compared with M/M groups (p?<?0.01). The conventional high-glucose culture media during primary culture of NRMCs induced ERS. We propose that medium-glucose culture media should be used and describe an improved protocol for the primary culture of NRMCs.     

Spatial and temporal population genetic variation and structure of Nothotsuga longibracteata (Pinaceae), a relic conifer species endemic to subtropical China

Nothotsuga longibracteata, a relic and endangered conifer species endemic to subtropical China, was studied for examining the spatial-temporal population genetic variation and structure to understand the historical biogeographical processes underlying the present geographical distribution. Ten populations were sampled over the entire natural range of the species for spatial analysis, while three key populations with large population sizes and varied age structure were selected for temporal analyses using both nuclear microsatellites (nSSR) and chloroplast microsatellites (cpSSR). A recent bottleneck was detected in the natural populations of N. longibracteata. The spatial genetic analysis showed significant population genetic differentiation across its total geographical range. Notwithstanding, the temporal genetic analysis revealed that the level of genetic diversity between different age class subpopulations remained constant over time. Eleven refugia of the Last Glacial Maximum were identified, which deserve particular attention for conservation management.