Characteristics of a mesoporous silicate immobilized trypsin bioreactor in organic media

Mesoporous silicates (MPS) materials are attractive materials for immobilizing proteins/enzymes because of their well ordered structures, large surface areas (up to 1000 m(2) g(-1)), narrow pore size distributions, large pore diameters, and pore volumes. MPS with average pore diameters ranging from 28 to 300 Angstroms were prepared using cationic and nonionic surfactants. The influence of water content, pH, storage, and thermal treatment on the activity of trypsin immobilized onto MPS was investigated. In a range of solvents, the amidolytic activity of immobilized trypsin was higher than that of the lyophilized preparation. Significant increases in k(cat)/K(M) occurred in propanol, ethanol, methanol, and formamide of 90, 62, 45 and 26, respectively. The observed increases were primarily a result of substantial increases in k(cat).     

Did backcrossing contribute to the origin of hybrid edible bananas?

Background

Bananas and plantains (Musa spp.) provide a staple food for many millions of people living in the humid tropics. The cultivated varieties (cultivars) are seedless parthenocarpic clones of which the origin remains unclear. Many are believed to be diploid and polyploid hybrids involving the A genome diploid M. acuminata and the B genome M. balbisiana, with the hybrid genomes consisting of a simple combination of the parental ones. Thus the genomic constitution of the diploids has been classified as AB, and that of the triploids as AAB or ABB. However, the morphology of many accessions is biased towards either the A or B phenotype and does not conform to predictions based on these genomic formulae.

Scope

On the basis of published cytotypes (mitochondrial and chloroplast genomes), we speculate here that the hybrid banana genomes are unbalanced with respect to the parental ones, and/or that inter-genome translocation chromosomes are relatively common. We hypothesize that the evolution under domestication of cultivated banana hybrids is more likely to have passed through an intermediate hybrid, which was then involved in a variety of backcrossing events. We present experimental data supporting our hypothesis and we propose a set of experimental approaches to test it, thereby indicating other possibilities for explaining some of the unbalanced genome expressions. Progress in this area would not only throw more light on the origin of one of the most important crops, but provide data of general relevance for the evolution under domestication of many other important clonal crops. At the same time, a complex origin of the cultivated banana hybrids would imply a reconsideration of current breeding strategies.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.     

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.     

Value at risk estimation using independent component analysis-generalized autoregressive conditional heteroscedasticity (ICA-GARCH) models

We suggest using independent component analysis (ICA) to decompose multivariate time series into statistically independent time series. Then, we propose to use ICA-GARCH models which are computationally efficient to estimate the multivariate volatilities. The experimental results show that the ICA-GARCH models are more effective than existing methods, including DCC, PCA-GARCH, and EWMA. We also apply the proposed models to compute value at risk (VaR) for risk management applications. The backtesting and the out-of-sample tests validate the performance of ICA-GARCH models for value at risk estimation.     

If you knew what was good for you! The value of environmental enrichments with known welfare benefits is not demonstrated by sows using operant techniques

This study assessed the motivation of gestating sows housed in standard, barren gestation stalls (used for breeding/implantation and/or gestation) for access to environmental enrichment. Enrichment consisted of a cotton rope or rubber mat in comparison to positive (additional food when fed at commercial levels) and negative (empty trough) controls. Although environmental enrichment may improve animal welfare, sows' valuation of enrichments is largely unknown. This study used an operant panel and obtained behavioral measures to quantify motivation. As indicated by a higher price paid and lower latencies to press the panel and enter the treatment stall (all comparisons, p < .05), sows demonstrated higher motivation for food compared with all treatments. Sows housed in gestation stalls did not demonstrate high motivation via operant responding for a cotton rope or a rubber mat; nor did they demonstrate any differences in behavioral measures (all comparisons, p > .10). Although sows' motivation for a mat did not differ from that for an empty trough, previous work has demonstrated the welfare benefits associated with comfort flooring.     

Precise Mapping of the CD95 Pre-Ligand Assembly Domain

Pre-association of CD95 at the plasma membrane is mandatory for efficient death receptor signaling. This homotrimerization occurs through self-association of an extracellular domain called the pre-ligand assembly domain (PLAD). Using novel molecular and cellular tools, we confirmed that CD95-PLAD is necessary to promote CD95 multimerization and plays a pivotal role in the transmission of apoptotic signals. However, while a human CD95 mutant deleted of the previously described PLAD domain (amino acids 1 to 66) fails to interact with its wild-type counterpart and trigger autonomous cell death, deletion of amino acids 1 to 42 does not prevent homo- or hetero (human/mouse)-oligomerization of CD95, and thus does not alter transmission of the apoptotic signal. Overall, these findings indicate that the region between amino acids 43 to 66 corresponds to the minimal motif involved in CD95 homotypic interaction and is necessary to convey an efficient apoptotic signal. Interfering with this PLAD may represent a new therapeutic strategy for altering CD95-induced apoptotic and non-apoptotic signals.     

Humoral immune responses to a single allele PfAMA1 vaccine in healthy malaria-naïve adults

Plasmodium falciparum: apical membrane antigen 1 (AMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. The polymorphic nature of AMA1 may compromise vaccine induced protection. The humoral response induced by two dosages (10 and 50 μg) of a single allele AMA1 antigen (FVO) formulated with Alhydrogel, Montanide ISA 720 or AS02 was investigated in 47 malaria-na?ve adult volunteers. Volunteers were vaccinated 3 times at 4 weekly intervals and serum samples obtained four weeks after the third immunization were analysed for (i) Antibody responses to various allelic variants, (ii) Domain specificity, (iii) Avidity, (iv) IgG subclass levels, by ELISA and (v) functionality of antibody responses by Growth Inhibition Assay (GIA). About half of the antibodies induced by vaccination cross reacted with heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of the antibody response, but had only a marginal influence on specificity, avidity, domain recognition or subclass responses. The highest antibody responses were observed for AMA1 formulated with AS02. The Growth Inhibition Assay activity of the antibodies was proportional to the amount of antigen specific IgG and the functional capacity of the antibodies was similar for heterologous AMA1-expressing laboratory strains. Trial registration: ClinicalTrials.gov NCT00730782.