Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.     

Bioclipse: an open source workbench for chemo- and bioinformatics

Background  

There is a need for software applications that provide users with a complete and extensible toolkit for chemo- and bioinformatics accessible from a single workbench. Commercial packages are expensive and closed source, hence they do not allow end users to modify algorithms and add custom functionality. Existing open source projects are more focused on providing a framework for integrating existing, separately installed bioinformatics packages, rather than providing user-friendly interfaces. No open source chemoinformatics workbench has previously been published, and no sucessful attempts have been made to integrate chemo- and bioinformatics into a single framework.     

Drug concentration-dependent expression of multidrug resistance-associated protein and P-glycoprotein in the doxorubicin-resistant acute myelogenous leukemia sublines.

The multidrug resistance of cancer cells can be mediated by an overexpression of the human MDR1 and MRP genes, which encode the transmembrane efflux pumps, the 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP), respectively. In this study, we investigate which protein is preferentially overexpressed in the function of doxorubicin concentrations in the acute myelogenous leukemia cell line (OCI/AML-2). Multidrug-resistant AML-2 sublines were isolated in doxorubicin concentrations of 20, 100, 250, and 500 ng/ml. MRP was at first expressed at low concentrations of less than 5 x IC50 (100 ng/ml) of doxorubicin followed by the overexpression of Pgp with concentrations of more than 12.5 x IC50 (250 ng/ml) of doxorubicin. In addition, it appeared that increased amounts of MRP and its mRNA in AML-2/DX20 and /DX100 decreased gradually in both AML-2/DX250 and /DX500 overexpressing Pgp. In conclusion, it is thought that the overexpression of MRP or Pgp is dependent upon drug concentrations. It could be implicated that the overexpression of MRP might be negatively related to that of Pgp.     

Fast evolutionary genetic differentiation during experimental colonizations

Founder effects during colonization of a novel environment are expected to change the genetic composition of populations, leading to differentiation between the colonizer population and its source population. Another expected outcome is differentiation among populations derived from repeated independent colonizations starting from the same source. We have previously detected significant founder effects affecting rate of laboratory adaptation among Drosophila subobscura laboratory populations derived from the wild. We also showed that during the first generations in the laboratory, considerable genetic differentiation occurs between foundations. The present study deepens that analysis, taking into account the natural sampling hierarchy of six foundations, derived from different locations, different years and from two samples in one of the years. We show that striking stochastic effects occur in the first two generations of laboratory culture, effects that produce immediate differentiation between foundations, independent of the source of origin and despite similarity among all founders. This divergence is probably due to powerful genetic sampling effects during the first few generations of culture in the novel laboratory environment, as a result of a significant drop in N _e. Changes in demography as well as high variance in reproductive success in the novel environment may contribute to the low values of N _e. This study shows that estimates of genetic differentiation between natural populations may be accurate when based on the initial samples collected in the wild, though considerable genetic differentiation may occur in the very first generations of evolution in a new, confined environment. Rapid and significant evolutionary changes can thus occur during the early generations of a founding event, both in the wild and under domestication, effects of interest for both scientific and conservation purposes.     

Incidence of Norovirus-Associated Medical Encounters among Active Duty United States Military Personnel and Their Dependents

BackgroundNorovirus is a leading cause of gastroenteritis episodes and outbreaks in US military deployments, but estimates of endemic disease burden among military personnel in garrison are lacking.MethodsDiagnostic codes from gastroenteritis-associated medical encounters of active duty military personnel and their beneficiaries from July 1998–June 2011 were obtained from the Armed Forces Health Surveillance Center. Using time-series regression models, cause-unspecified encounters were modeled as a function of encounters for specific enteropathogens. Model residuals (representing unexplained encounters) were used to estimate norovirus-attributable medical encounters. Incidence rates were calculated using population data for both active duty and beneficiary populations.ResultsThe estimated annual mean rate of norovirus-associated medically-attended visits among active duty personnel and their beneficiaries was 292 (95% CI: 258 to 326) and 93 (95% CI: 80 to 105) encounters per 10,000 persons, respectively. Rates were highest among beneficiaries <5 years of age with a median annual rate of 435 (range: 318 to 646) encounters per 10,000 children. Norovirus was estimated to cause 31% and 27% of all-cause gastroenteritis encounters in the active duty and beneficiary populations, respectively, with over 60% occurring between November and April. There was no evidence of any lag effect where norovirus disease occurred in one population before the other, or in one beneficiary age group before the others.ConclusionsNorovirus is a major cause of medically-attended gastroenteritis among non-deployed US military active duty members as well as in their beneficiaries.     

Overexpression of Class III Beta Tubulin and Amplified HER2 Gene Predict Good Response to Paclitaxel and Trastuzumab Therapy

Through this study, we aimed to validate several biomarkers that have been known to possibly predict the outcomes of the trastuzumab and paclitaxel (TP). Human epidermal growth factor 2 (HER2) positive metastatic breast cancer (MBC) patients who had been treated with TP in single institute from 2006 to 2009 were included in this study. For procured formalin fixed paraffin embedded tumor tissues, HER2 amplification index (AI) and polymorphisms of the immunoglobulin G fragment C receptors (FCGR) were assessed as biomarkers to the trastuzumab and expression of class III beta tubulin (bTubIII) was evaluated as a predictive factor to the paclitaxel. Of 46 patients treated with TP, 27 patients could be evaluated for HER2 AI, 31 for bTubIII, and 26 for FCGR gene polymorphism. The median of the HER2 AI was 5.0 (range, 1.4−15.5) and a higher HER2 AI (≥5.0) was significantly correlated with better response rate (RR) (80% vs. 42%, P = 0.049) and longer progression-free survival (PFS) (13.6 vs. 6.9 months, P = 0.023). High bTubIII expression showed higher RRs than did low expression (81% vs. 40%, P = 0.040) in addition to longer PFS (16.2 months vs. 8.8 months, P = 0.04). However, polymorphisms in FCGR 2A-H131R or FCGR 3A-V158F were not predictive of RR or PFS. Our results suggest that a high HER2 AI and high bTubIII expression could be predictive of the outcomes to TP therapy but no evidence was found in terms of FCGR polymorphisms.     

Improved detergent-based recovery of polyhydroxyalkanoates (PHAs)

Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process.