Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

During attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons revealed that the genome sequence of the new virus was 91-93% identical to those of known murine hepatitis viruses (MHVs). The predicted open reading frame from the nucleotide sequence encoded all known proteins of MHVs. Analysis at the protein level showed that the virus was closely related to the highly virulent MHV-JHM strain. The virus strain was named MHV-MI. No type D retroviruses were found. Degenerate PCR targeting of type D retrovirus and 5'-RACE targeting of other types of retroviruses confirmed the absence of any retroviral association with the LCH xenografted SCID mice.

Improvement of odor intensity measurement using dynamic olfactometry.

Odor intensity reveals a dose-effect relationship between inhaled odor and perceived odor sensation by the receptors, while odor concentration reflects the odor strength at the emission sources. The study reports significant improvements in experimental procedures in establishing the odor concentration-intensity (OCI) relationships using a newly developed digital olfactometer. The improvements in experimental procedures have been made to meet the requirements of both the VDI guideline 3882.1 and the European standard (EN13725). Several areas which could affect the reliability of the results have been identified in some similar studies. The latest digital olfactometer was calibrated automatically to ensure accurate and repeatable dilution ratios. Cross contamination has been eliminated through the instrument design and extensive cleaning procedures, making random presentation possible. Stringent panelist screening and continuous performance monitoring ensures consistent sensitivity of the panel. The extension of odor intensity category to temperature sensation gives a reference to assist judgments of perceived odor sensation. The DynaScent calculation method has simplified odor intensity calculation and can be applied to many odor samples. A total of 38 odor samples from three alumina refinery sites and two sewage treatment plants were collected for analysis. The results have confirmed the efficiency of the olfactometer. Distinct Odor Concentrations (DOCs) were calculated for each sample using both VDI and DynaScent methods. A student t test on two major odor types confirmed that there are no significant differences between two methods. The study has shown the DOCs for refinery odor and wastewater odor are in the range of 3.8-15.4 and 4.2-15.6 odor unit (OU)/m3 respectively. The study demonstrated that the improvements are critical in achieving reliable odor intensity measurement. This can lead to the setup of quantitative odor impact criteria for different industries and sites.

Mutation detection using mass spectrometric separation of tiny oligonucleotide fragments.

A DNA mutation detection protocol able to identify and characterize a previously unknown change in a given sequence in a rapid, efficient, sensitive, and inexpensive manner is required to take advantage of the resources now available to researchers through the genome sequencing projects. We have developed a method based on base-specific cleavage of polymerase chain reaction (PCR) products and then separation of the fragments by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), which can meet these criteria. Differences are seen as the presence, absence, or mass change of peaks corresponding to fragments affected by the base difference. This technique is shown through the detection of a polymorphism in the 3' untranslated region of IL12p40 from a double-stranded PCR product, and the detection of a single nucleotide polymorphism between two mouse strains. The sensitivity of the technique can be increased with the use of postsource decay, which enables differentiation of two fragments of identical mass but different sequence. The level of specificity and the rapid sample analysis time lend this technique to the mass screening of individuals for sequence changes and, in combination with MS sequencing methods, could be used to facilitate rapid resequencing of DNA.