Automated topographical cell proliferation analysis

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.     

The devastating outcome of massive subcutaneous injection of highly viscous fluids in male-to-female transsexuals

Illicit subcutaneous injections of massive quantities of highly viscous fluids are still performed, often by unqualified persons. Fifteen male-to-female transsexuals consulted the authors regarding their devastating long-term outcomes after the injection of up to 8 liters of alleged silicone or mineral oil to feminize their bodies. After a latency period of up to 17 years, these injections led to complications ranging from scarring and deformity to infections. These patients were treated conservatively for inflammation and infection or surgically by resection of the oil-infested areas. In view of the potential dangers, feminization by the injection of high-viscosity fluids should be soundly condemned.     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

Reliability of a structured method of selecting abstracts for a plastic surgical scientific meeting

There is no generally accepted method for assessing abstracts that are submitted for a medical scientific meeting. This article describes the development and prospective evaluation of such a method applied to the 220 abstracts submitted for the 2000 Annual Meeting of the European Association of Plastic Surgeons. Structured abstracts were evaluated in three categories: aesthetic surgery, basic research, and clinical study. Each anonymous abstract was assessed separately by 10 reputable European plastic surgeons. These reviewers used a structured rating questionnaire which resulted in a score given by each reviewer to each abstract between -6 and +6. The scores of all 10 reviewers were added for each abstract, and the papers were accepted in each of the three categories on the basis of this abridged score. To evaluate the reliability of this structured method of selection, the interrater agreement among the reviewers was tested by means of kappa analysis and the Cronbach alpha coefficient. The kappa values for agreement among reviewers regarding acceptability of abstracts were low, but the alpha coefficient indicated an acceptable degree of reliability of the average reviewers' ratings for all categories. Using a structured questionnaire can be helpful in the objective assessment of abstracts for a scientific meeting and may facilitate comparison of abstracts. Meritocratic dichotomy of abstracts by the reviewers is advocated to further improve reliability of the rating. Even though reliability generally increases with the number of reviewers, the annual increase of submitted abstracts may necessitate a decrease in the number of reviewers for each abstract.     

Ruthenium(II)-phenanthroline-biotin complexes: synthesis and luminescence enhancement upon binding to avidin

We report the synthesis, characterization, and avidin-binding properties of two novel ruthenium complexes, [Ru(bpy)(2)(phen-biotin)][PF(6)](2) 1 and [Ru(phen)(2)(phen-biotin)][PF(6)](2) 2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline, phen-biotin = 5-(10-amidobiotinyl)-1,10-phenanthroline)). We demonstrate that both biotinylated compounds bind to avidin through their biotin moieties with high affinity and in a 4:1 ratio. The binding of compounds 1 and 2 to avidin results in an enhancement in luminescence intensity ( approximately 1.4x, approximately 1.6x, respectively), relative to the unbound biotinylated ruthenium complexes. This behavior is markedly different from biotinylated organic dyes, whose fluorescence is quenched upon binding to avidin. Thus, ruthenium-biotin complexes 1 and 2 can form the basis of new, simplified biotin-avidin assays, which involve luminescence detection of the relevant biotinylated molecule through cross-linking with avidin.     

History offhand: a golfer's needleholder

Sir Harold D. Gillies was the father of modern plastic surgery. Apart from being an extraordinary surgeon, he was an innovator of surgical instruments and an outstanding golfer. He adopted Kocher's suture scissors and converted them for needleholding. The authors believe that the surgeon Gillies adapted this needleholder to fit his golfer's hand.     

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aβ peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase.