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%.     

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.     

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.     

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.     

Reduced Regional Homogeneity in Bilateral Frontostriatal System Relates to Higher Impulsivity Behavior in Codeine-Containing Cough Syrups Dependent Individuals

Background

In the past twenty years, codeine-containing cough syrups (CCS) was recognized as a new type of addictive drugs. However, the exact neurobiologic mechanisms underlying CCS-dependence are still ill-defined. The aims of this study are to identify CCS-related modulations of neural activity during the resting-state in CCS-dependent individuals and to investigate whether these changes of neural activity can be related to duration of CCS use, the first age of CCS use and impulse control deficits in CCS-dependent individuals. We also want to observe the impact of gray matter deficits on these functional results.

Methodology/Principal Findings

Thirty CCS-dependent individuals and 30 control subjects participated. Resting-state functional MRI was performed by using gradient-echo echo-planar imaging sequence. Regional homogeneity (ReHo) was calculated by using REST software. Voxel-based analysis of the ReHo maps between controls and CCS-dependent groups was performed using two-sample t tests (p<0.05, corrected for multiple comparisons). The Barratt Impulsiveness Scale 11 (BIS.11) was surveyed to assess participants'' impulsivity trait soon after MR examination. Abnormal clusters revealed by group comparison were extracted and correlated with impulsivity, duration of CCS use, and age of first CCS use. ReHo was diminished in the bilateral medial orbitofrontal cortex (mOFC) and left dorsal striatum in CCS-dependent individuals. There were negative correlations between mean ReHo in the bilateral medial OFC, left dorsal striatum and duration of CCS use, BIS.11 total scores, and the subscale of attentional impulsivity in CCS-dependent individuals. There was a significantly positive correlation between mean ReHo in the left dorsal striatum and age of first CCS use in CCS-dependent individuals. Importantly, these results still remain significant after statistically controlling for the regional gray matter deficits.

Conclusion

Resting-state abnormalities in CCS-dependent individuals revealed in the present study may further improve our understanding about the neural substrates of impulse control dysfunction in CCS-dependent individuals.     

GNB3, eNOS,and Mitochondrial DNA Polymorphisms Correlate to Natural Longevity in a Xinjiang Uygur Population

Background

In centenarian populations, application of the positive biology approach (examination of positive phenotypes in aging) has revealed that mitochondrial DNA (mtDNA) mutation accumulation may be linked to human longevity; however, the role of guanine nucleotide-binding protein (G protein) abnormalities modulated by G-protein beta-3 (GNB3) and nitrate (NO2) production associated with endothelial nitric oxide synthase (eNOS), commonly appearing in age-related diseases, remains undetermined.

Objective

The association between the mtDNA 5178A/C, mtDNA 10398A/G, GNB3 C825T, and eNOS polymorphisms and longevity in a Uygur population (Xinjiang region, China) were investigated.

Methods

A total of 275 experimental subjects aged ≥100 or with 4 generations currently living were screened for inclusion in the centenarian (>100 years) and nonagenarian groups (90–100 years), and 112 65–70 year old control subjects were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to examine mtDNA 5178A/C, mtDNA 10398A/G, GNB3 C825T, and eNOS. Associations between polymorphic loci, genotypes, and longevity were analyzed.

Results

165 included subjects (M∶F = 107∶58; mean age = 97±3 years; mean age 100–113 years) were assigned to the centenarian (M∶F = 46/19; n = 65) and nonagenarian groups (M∶F = 61/39; n = 100). Associations between mtDNA C5178A and A10398G polymorphisms with longevity in the centenarian group with mtDNA genotype frequencies 5178A and 10398G were 66.79% and 36.8%.

Conclusions

Applying the overwhelming longevity observed in Uygur populations, these findings demonstrate that mtDNA 5178A/C and 10398A/G, GNB3 C825T, and eNOS polymorphisms are useful as a genetic basis for longevity.     

Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.     

Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.