Discrimination of Cricotopus species (Diptera: Chironomidae) by DNA barcoding.

Chironomids (Diptera) typically comprise the most abundant group of macroinvertebrates collected in water quality surveys. Species in the genus Cricotopus display a wide range of tolerance for manmade pollutants, making them excellent bioindicators. Unfortunately, the usefulness of Cricotopus is overshadowed by the difficulty of accurately identifying larvae using current morphological keys. Molecular approaches are now being used for identification and taxonomic resolution in many animal taxa. In this study, a sequence-based approach for the mitochondrial gene, cytochrome oxidase I (COI), was developed to facilitate identification of Cricotopus species collected from Baltimore area streams. Using unique COI sequence variations, we developed profiles for seven described Cricotopus sp., four described Orthocladius sp., one described Paratrichocladius sp. and one putative species of Cricotopus. In addition to providing an accurate method for identification of Cricotopus, this method will make a useful contribution to the development of keys for Nearctic Cricotopus.

BRCA1 and BRCA2 have a limited role in familial prostate cancer.

Epidemiological studies have suggested that the breast cancer susceptibility genes, BRCA1 and BRCA2, may be involved in the development of prostate cancer. Several studies have screened prostate cancer populations for the presence of BRCA1 and BRCA2 mutations, with few mutations identified. In this study, 22 high-risk prostate cancer families (at least three cases of prostate cancer) were screened by conformation-sensitive gel electrophoresis (CSGE) for mutations in BRCA1 and BRCA2. To maximize the chance of finding mutations in these two genes, families were also selected for the presence of at least two cases of breast and/or ovarian cancer. We identified one previously reported BRCA2 missense mutation and two previously unreported BRCA2 intron polymorphisms. No BRCA1 or BRCA2 truncating mutations were detected. Thus, BRCA1 and BRCA2 appear to have a limited role in familial prostate cancer, and families with both prostate and breast cancer may result from mutations in other predisposition genes.

17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGma1B genes.

Amplification of the 17q23 region occurs frequently in breast tumors. To characterize the structure of 17q23 amplicons and to identify oncogene targets associated with this alteration, we performed a copy number analysis of 87 17q23 localized expressed sequence tags in seven breast cancer cell lines. Three major regions of amplification were detected in the MCF7 and BT474 cell lines. Amplification of at least one of four known genes (PAT1, PS6K, RAD51C, and SIGMA1B) was detected in the cell lines and in 28% of 94 breast tumors. In most cases, these four genes were overexpressed when amplified, but there was a particularly good association between amplification of the SIGMA1B gene and elevated expression in tumors, which suggested a possible role for this gene in tumor progression. Our data show that this region contains at least four independent targets of amplification, which suggests that there is considerable variability in the structure of the 17q23 amplicon.

Design, development and qualification of a microbiological challenge facility to assess the effectiveness of BFS aseptic processing.

A programme of work has been initiated to further the understanding of the impact of the environment surrounding a Blow/Fill/Seal (BFS) machine upon the microbiological quality of processed product. A dedicated facility (Challenge Room) has been constructed and qualified to provide for the production and containment of dispersions of micro organisms in air of a room housing an operating BFS machine of a given style and configuration. The facility achieves homogeneous distribution of generated dispersions throughout the Challenge Room air, including that within and close to the critical area in which aseptic BFS operations occur. Generated microbial dispersions can be maintained in the room over extended time periods (up to 600 min) at a desired concentration within the range 10(1) to 10(7) cfu m(-3). They can also be produced employing different cell types, including bacterial endospores, Gram-positive and Gram-negative vegetative cells and yeast cells. Effective containment of dispersions is achieved while 'cards of product' (vials in sets) are conveyed from the Challenge Room to an adjacent Packing and Storage Area. Decontamination of the room and the housed BFS machine is accomplished through exposure to chlorine dioxide gas at a concentration of 1.0 mg dm(-3) for 120 min at room temperature (approximately 23 degrees C).

Performance of blow/fill/seal equipment under controlled airborne microbial challenges.

Fundamental investigations have been carried out into the effectiveness of aseptic fill using Blow/Fill/Seal machinery. Techniques have been developed to generate and to maintain over prolonged periods, controlled microbial challenges of spores of Bacillus subtilis var. niger dispersed within the air of a containment room housing a Blow/Fill/Seal machine set-up to undertake medium fill studies. The range of spore concentrations that have been generated extends from as low as 3 x 10(2) to as high as 10(7) spores m-3. Responses to controlled microbial challenges have defined the relationship between product contamination and microbiological quality of the machine environment. The relationship is regular and amenable to extrapolation, so that it is now practicable to specify the microbiological quality of the machine environment, together with machine operating conditions, needed to attain a Sterility Assurance Level comparable to that targeted for terminal sterilization (i.e. 10(-6)). The impacts of mould configuration, air shower operation and location of point of fill on product contamination have also been examined.

Drapes as barriers to airborne bacterial spores.

The primary function of a package enclosing a sterile medical or surgical device (e.g. syringe, needle, scalpel, etc) or a tray wrap enveloping a set of surgical items is to be an absolute barrier towards micro-organisms. Generally, the barrier function operates from the time that the item is exposed to the sterlization treatment until it is used. It follows that the material(s) constituting the package or wrap must permit the sterilization treatment to perform effectively, yet present a barrier to potential microbiological contaminants during post sterlization storage, handling and transport.

Airborne microbial challenges of Blow/Fill/Seal equipment: a case study.

Controlled microbial challenges, comprising air-dispersed spores of Bacillus subtilis var niger, have been generated within a containment room (around 54 m3 in volume) housing a Blow/Fill/Seal machine. 'Stirred-settling' conditions were created throughout the room and the airborne spore challenge was monitored to ensure homogeneity within the room for extended periods of time. The Blow/Fill/Seal machine was set to fill 2 cm3 ampoules with Tryptone Soya Broth at each of three airborne challenge levels of 10(4), 10(6) and 10(7) spores m-3 (about 10(2), 10(4) and 10(5) spores ft-3 respectively). A relationship has been established between the level of airborne micro-organisms in the machine operating environment and the extent of product contamination. This relationship allows prediction of operating conditions under which a level of sterility assurance, equal to that demanded of terminal sterilization, is attained. It is stressed that the findings apply only to the particular Blow/Fill/Seal machine and to the specific conditions of machine operation.

Comparative genomic sequence analysis and isolation of human and mouse alternative EGFR transcripts encoding truncated receptor isoforms.

This study presents the annotated genomic sequence and exon-intron organization of the human and mouse epidermal growth factor receptor (EGFR) genes located on chromosomes 7p11.2 and 11, respectively. We report that the EGFR gene spans nearly 200 kb and that the full-length 170-kDa EGFR is encoded by 28 exons. In addition, we have identified two human and two mouse alternative EGFR transcripts of 2.4-3.0 kb using both computational and experimental methods. The human 3.0-kb and mouse 2.8-kb EGFR mRNAs are predominantly expressed in placenta and liver, respectively, and both transcripts encode 110-kDa truncated receptor isoforms containing only the extracellular ligand-binding domain. We also have demonstrated that the aberrant 2.8-kb EGFR transcript produced by the human A431 carcinoma cell line is generated by splicing to a recombinant 3'-terminal exon located in EGFR intron 16, which apparently was formed as a result of a chromosomal translocation. Finally, we have shown that the human, mouse, rat, and chicken 1.8- to 3.0-kb alternative EGFR transcripts are generated by distinct splicing mechanisms and that each of these mRNAs contains unique 3' sequences that are not evolutionarily conserved. The presence of truncated receptor isoforms in diverse species suggests that these proteins may have important functional roles in regulating EGFR activity.