A novel DNAseq program for enhanced analysis of Illumina GAII data: a case study on antibody complementarity-determining regions.

High-throughput DNA sequencing technologies are increasingly becoming powerful systems for the comprehensive analysis of variations in whole genomes or various DNA libraries. As they are capable of producing massive collections of short sequences with varying lengths, a major challenge is how to turn these reads into biologically meaningful information. The first stage is to assemble the short reads into longer sequences through an in silico process. However, currently available software/programs allow only the assembly of abundant sequences, which apparently results in the loss of highly variable (or rare) sequences or creates artefact assemblies. In this paper, we describe a novel program (DNAseq) that is capable of assembling highly variable sequences and displaying them directly for phylogenetic analysis. In addition, this program is Microsoft Windows-based and runs by a normal PC with 700MB RAM for a general use. We have applied it to analyse a human naive single-chain antibody (scFv) library, comprehensively revealing the diversity of antibody variable complementarity-determining regions (CDRs) and their families. Although only a scFv library was exemplified here, we envisage that this program could be applicable to other genome libraries.

Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody.

Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO(3)H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds.

Comparison of two radio-frequency plasma sterilization processes using microspot evaluation of microbial inactivation.

In this study, we evaluated gas plasma surface sterilization methods in a specific sterilizer. We have introduced a new monitoring method using 0.4 microm pore size membranes, which in this study gave the information corresponding to 3000 exposed biological indicators per treatment cycle. This enabled us to compare the fraction of inoculates that showed no growth after exposure for 30 different locations in the chamber, and hereby identify weak and strong spots in the chamber with regard to sporicidal effect. Membranes were also used to expose a broad spectrum of soil bacteria for plasma treatment at four different conditions. The organisms were identified using PCR and sequencing. The test showed that Bacillus stearothermophilus spores were inactivated at the slowest rate among the tested microorganisms. Further alpha-proteobacteria (Gram negative) seemed more sensitive than the rest of the tested organisms. The microspot evaluation approach has been a most useful tool in the assessment of sterilization performance in sterilizers that do not have clear measurable parameters related to the sterilization.

The dependence of the sporicidal effects on the power and pressure of RF-generated plasma processes.

The sporicidal effect of 20 different radio-frequency plasma processes produced by combining five different gas mixtures [O(2), Ar/H(2) (50/50%), Ar/H(2) (5/95%), O(2)/H(2) (50/50%), O(2)/H(2) (95/5%)] with four power/pressure settings were tested. Sporicidal effects of oxygen-containing plasmas were dependent on power at low pressure settings but not at high pressure settings. In the absence of oxygen no power dependency was observed at either high or low pressure settings. Survivor curves obtained with the use of nonoxygen plasmas typically had a tailing tendency. Only a mixture-optimized Ar/H(2) (15/85%) plasma process was not encumbered by tailing, and produced a decimal reduction time (D value) below 2 min for Bacillus stearothermophilus spores. Scanning electron microscopy showed that a CF(4)/O(2) plasma did more damage to the substrate than the 15/85% Ar/H(2) plasma. The present results indicate that UV irradiation inactivation is swift and power and pressure independent. Additionally, it is produced at low energy. However, it is not complete. Inactivation through etching is highly power and pressure dependent; finally, inactivation by photodesorption is moderately power and pressure dependent. A sterilization process relying on this mechanism is very advantageous because it combines a highly sporicidal effect with low substrate damage.

Optimization of a RF-generated CF4/O2 gas plasma sterilization process.

A sterilization process with the use of RF-generated (13.56 MHz) CF(4)/O(2) gas plasma was optimized in regards to power, flow rate, exposure time, and RF-system type. The dependency of the sporicidal effect on the spore inoculum positioning in the chamber of the RF systems was also investigated. Dried Bacillus stearothermophilus ATCC 7953 endospores were used as test organisms. The treatments were evaluated on the basis of survival curves and corresponding D values. The only parameter found to affect the sterilization process was the power of the RF system. Higher power resulted in higher kill. Finally, when the samples were placed more than 3-8 cm away from a centrally placed electrode in System 2, the sporicidal effect was reduced. The results are discussed and compared to results from the present literature. The RF excitation source is evaluated to be more appropriate for sterilization processes than the MW source.