Applied Biochemistry and Biotechnology (v.135, #2)

The statistical design of experiments (DOE) is a collection of predetermined settings of the process variables of interest, which provides an efficient procedure for planning experiments. Experiments on biological processes typically produce long sequences of successive observations on each experimental unit (plant, animal, bioreactor, fermenter, or flask) in response to several treatments (combination of factors). Cell culture and other biotech-related experiments used to be performed by repeated-measures method of experimental design coupled with different levels of several process factors to investigate dynamic biological process. Data collected from this design can be analyzed by several kinds of general linear model (GLM) statistical methods such as multivariate analysis of variance (MANOVA), univariate ANOVA (timesplit-plot analysis with randomization restriction), and analysis of orthogonal polynomial contrasts of repeated factor (linear coefficient analysis). Last, regression model was introduced to describe responses over time to the different treatments along with model residual analysis. Statistical analysis of biprocess with repeated measurements can help investigate environmental factors and effects affecting physiological and bioprocesses in analyzing and optimizing biotechnology production.
Keywords: Statistical experimental design; repeated-measures (RM); empirical (statistical) model; dynamic bioprocess; sphericity

Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e. they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18,2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.
Keywords: Citric acid; cellulases; protoplast fusionTrichoderma spp.; Aspergillus niger

Electrolyzed-reduced water protects against oxidative damage to DNA, RNA, and protein by Mi Young Lee; Yoon Kyoung Kim; Kun Kul Ryoo; Yoon Bae Lee; Eun Ju Park (133-144).
The generation of reactive oxygen species is thought to cause extensive oxidative damage to various biomolecules such as DNA, RNA, and protein. In this study, the preventive, suppressive, and protective effects of in vitro supplementation with electrolyzed-reduced water on H2O2-induced DNA damage in human lymphocytes were examined using a comet assay. Pretreatment, cotreatment, and posttreatment with electrolyzed-reduced water enhanced human lymphocyte resistance to the DNA strand breaks induced by H2O2 in vitro. Moreover, electrolyzed-reduced water was much more effective than diethylpyrocarbonate-treated water in preventing total RNA degradation at 4 and 25°C. In addition, electrolyzed-reduced water completely prevented the oxidative cleavage of horseradish peroxidase, as determined using sodium dodecyl sulfate-polyacrylamide gels. Enhancement of the antioxidant activity of ascorbic acid dissolved in electrolyzed-reduced water was about threefold that of ascorbic acid dissolved in nonelectrolyzed deionized water, as measured by a xanthine-xanthine oxidase superoxide scavenging assay system, suggesting an inhibitory effect of electrolyzed-reduced water on the oxidation of ascorbic acid.
Keywords: Electrolyzed-reduced water; oxidative damage; DNA; RNA; protein

Functional analysis of type II thioesterase of Streptomyces lydicus AS 4.2501 by Feng-Ming Yu; Bin Qiao; Feng Zhu; Jin-Chuan Wu; Ying-Jin Yuan (145-158).
Constructing a mutant strain of single gene disruption is the basis for the study of gene function and metabolomics. Systematic and complete genome sequencing is the basis of genetic manipulation. In the case of a little knowledge about the Streptomyces lydicus genome and the speculation that polyketide synthases (type I) might be responsible for the polyketide side chain biosynthesis of streptolydigin, a 588-bp fragment was amplified by polymerase chain reaction (PCR) according to the homology existing in the same functional genes among Streptomyces. A mutant strain of this gene was constructed by single crossover homologous recombination. The results of sequence analysis as well as the metabolite analysis of the mutant and the original strain by liquid chromatography/mass spectroscopy indicated that this fragment was part of type II thioesterase (TE) gene, which was required for streptolydigin biosynthesis like other type II TEs function in related antibiotics biosynthesis. Furthermore, targeted gene manipulation based on PCR was a powerful tool for studying gene function and metabolomics, especially when little was known about the genomic sequence of streptomyces.
Keywords: Type II thioesterase; polymerase chain reaction; mutant strain construction; functional analysis; single crossover homologous recombination; Streptomyces lydicus AS 4.2501

Differential response induced by exposure to low-dose ionizing radiation in SHSY-5Y and normal human fibroblast cells by A. McLachlan-Burgess; S. McCarthy; C. Griffin; J. Richer; R. G. Cutler; S. Pandey (159-177).
Radiation therapy has been used in the treatment of a wide variety of cancers for nearly a century and is one of the most effective ways to treat cancer. Low-dose ionizing radiation (IR) can interfere with cell division of cancer and normal cells by introducing oxidative stress and injury to DNA. The differences in the response to IR-induced DNA damage and increased reactive oxygen species between normal human fibroblasts (NHFs) and cancerous SHSY-5Y cells were considered. H2AX staining and comet assays revealed that NHF cells responded by initiating a DNA repair sequence whereas SHSY-5Y cells did not. In addition, NHF cells appeared to quench the oxidative stress induced by IR, and after 24 h no DNA damage was present. SHSY-5Y cells, however, did not repair their DNA, did not quench the oxidative stress, and showed characteristic signs that they were beginning to undergo apoptosis. These results indicate that there is a differential response between this cancerous and normal cell line in their ability to respond to low-dose IR, and these differences need to be exploited in order to treat cancer effectively. Further study is needed in order to elucidate the mechanism by which SHSY-5Y cells undergo apoptosis following radiation and why these normal cells are better equipped to deal with IR-induced double-strand breaks and oxidative stress.
Keywords: Radiation; apoptosis; double-strand breaks; DNA damage; DNA repair

Erratum to: Optimization of an innovative hollow-fiber process to produce lactose-reduced skim milk by Winfried Neuhaus; Senad Novalin; Mario Klimacek; Barbara Splechtna; Inge Petzelbauer; Alexander Szivak; Klaus D. Kulbe (179-179).