A systematic approach to identifying urothelial cells likely to be polysomic by fluorescence in situ hybridization

OBJECTIVE: To determine which nuclear morphologic and background features are most associated with normal and abnormal chromosome patterns in urine cells evaluated by fluorescence in situ hybridization (FISH) with Vysis UroVysion (Downers Grove, Illinois, USA). STUDY DESIGN: One hundred specimens were analyzed and the nuclear morphologic and background features compared between FISH-negative (disomic) and -positive (polysomic) cases. RESULTS: Our data show that polysomic urothelial cells have significantly (p < 0.001) weaker DAPI nuclear counterstaining, nuclear enlargement, more irregular nuclear shape and more chromatin clumping when compared to disomic cells. CONCLUSION: These findings indicate that there are specific staining and nuclear cytologic features that can help identify cells that have a high probability of being polysomic by FISH. This information may help cytologists decrease the time required to evaluate urine specimens by FISH and may aid in the development of instruments that automate FISH analysis.     

Activity of immobilized papain dehydrated by n-propanol in low-water media

A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min(-1) (mg powder)(-1) in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 degrees C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.     

The importance of bead size measurement in mass-transfer modelling with immobilised cells

The effective diffusivity of O₂ inside immobilised cell particles has been much discussed. Most reported estimates are based on fitting a mass-transfer reaction model to measured total oxygen uptake rates. The particle diameter has the largest single influence in such models, but its accurate measurement has probably recieved insufficient attention. We have studied sorbitol and glucose oxidation by cells of Gluconobacter suboxydans entrapped in calcium alginate gel beads. These beads were found to shrink rapidly in air, so that size measurement under water is essential. By comparison with rigid particles of similar known size, it was shown that measurement of the microscopic image gives a systematic underestimate. In consequence, the fitted oxygen diffusivity will be around 20% too low. Careful attention to size measurement gave good agreement between diffusivity estimates from beads with different mean sizes and cell loadings, with a best value of 2.51 × 10⁹ m²s^–1, 92% of the value for pure water. The estimated diffusivity is not significantly affected by a distribution of bead sizes with up to 10% standard deviation about the same mean.     

Biocatalysis in low-water media: understanding effects of reaction conditions

The key role played by counter-ions with enzymes in low-water systems has become better appreciated with, for example, large effects on enantioselectivity. In low-dielectric media, counter-ions will associate strongly with charges in the protein or its substrates. Studies of temperature dependence have shown that hard-to-model entropies have a significant effect on behaviour, including enantioselectivity. Evidence has been presented that the supramolecular organisation of enzyme molecules can have important effects on behaviour, for example collapse of microstructure in cross-linked crystals.     

FISHing for pancreatobiliary tract malignancy in endoscopic brushings enhances the sensitivity of routine cytology

Pancreatobiliary tract carcinoma is a lethal disease with low survival rates and limited treatment options. Diagnosis is complicated by benign conditions that can mimic malignancy on radiological studies (e.g. primary sclerosing cholangitis or PSC) and the suboptimal sensitivity of endoscopic biopsy/brushings obtained by endoscopic retrograde cholangiopancreatography (ERCP). The detection of multiple chromosomal gains by fluorescence in situ hybridization (FISH), referred to as polysomy, has demonstrated improved sensitivity over routine cytological evaluation. The evaluation of brushings by both routine cytology and FISH in our cytopathology laboratory has been in clinical practice since 2003. Strong morphological and screening skills enable cytotechnologists to become proficient in the assessment of FISH slides, which translates into cost and time savings. Multiple reports from various institutions have demonstrated the utility of FISH for patients with and without PSC. The incorporation of routine cytology and FISH results into the management algorithm for patients under suspicion for pancreatobiliary malignancy is a testament to the clinical success of these cytological assays.     

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.     

Symmetry indices based on accelerometric data in trotting horses

Detection and quantification of lameness in horses consists primarily of a subjective assessment, whereby both intra- and inter-observer disagreements exist, especially with low grade lameness. Therefore, clinically applicable methods are needed for reliable, objective assessments. The aim of this study was to describe three symmetry indices derived from a simple accelerometric method and investigate these in sound trotting horses. The indices describe the overall symmetry of the gait, the symmetry of loads placed on the limbs and the symmetry in timing between left and right steps. These symmetry indices were able to quantify the high degree of symmetry of the trot in sound horses that has been described in earlier studies using other gait analysis methods. Also, we have analysed the variances and have found high repeatability for all three indices.This provides a basis for future investigations of the symmetry indices and their potential for objective detection and quantification of lameness in horses.     

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1.

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.     

Dietary carbohydrate dictates development of Type 2 diabetes in the Nile rat

Amount and type of dietary carbohydrate (CHO), as well as the CHO:fat ratio, are thought to be critical for both the rate of development and severity of Type 2 diabetes mellitus. Thus, these nutritional considerations were examined in the previously described “spontaneous” model of diabetes and metabolic syndrome, the Nile rat. Weanling male Nile rats (n=92) were fed semipurified diets, modifying glycemic index and load by changing the amount of fiber or altering the CHO:fat ratio. Random and fasting blood glucose and body weight were assessed, and diabetes was characterized in terms of blood glucose, relevant plasma and liver parameters, food and water intake and terminal organ weights. Nile rats fed with hiCHO became more hyperglycemic than rats fed with modCHO (P<.05), while loCHO and hiCHO+hiFiber rats remained essentially normoglycemic. Liver lipid and glycogen accumulation was associated with severe hyperlipemia in diabetic rats, analogous to metabolic syndrome in humans. Advanced diabetes was linked to liver and kidney damage and elevated blood urea nitrogen with weight loss. Dispersing dietary CHO by fiber or replacing it by moderate fat (reducing the glycemic index and load) delayed the onset of diabetes but did not prevent signs of insulin resistance. A very low content of dietary CHO (high fat) seemed to prevent even these early indicators of insulin resistance. Thus, the Nile rat represents a novel CHO-sensitive model for study of Type 2 diabetes that reliably follows the course of disease in humans.