Backbone and side-chain assignment of the lipidated and non-lipidated forms of the meningococcal outer membrane protein LP2086.

LP2086 is a lipidated outer membrane protein from Neisseria meningitidis that elicits bactericidal antibodies and represents a promising vaccine candidate against meningococcal infections. Here we report the backbone and side-chain assignment for two forms of LP2086: non-lipidated in aqueous buffer and the lipidated protein in micellar solution.

Phase I clinical trials of aroA aroD and aroA aroD htrA attenuated S. typhi vaccines; effect of formulation on safety and immunogenicity.

PBCC211, an aroA aroD derivative of S. typhi strain CDC10-80, was tested in phase I trials as a single dose typhoid fever vaccine. Three different vaccine preparations, reconstituted lyophilized bacteria, freshly grown bacteria or lyophilized bacteria reconstituted from sachets, were orally administered to a total of 86 adult volunteers. An aroA aroD htrA strain, PBCC222, was also tested in 38 volunteers. Formulation impacted on the determination of a safe and immunogenic dose; reconstituted lyophilized cultures required higher doses than the broth cultures to stimulate seroconversion. In general, doses which seroconverted the majority of group members produced undesirable symptoms regardless of attenuation or formulation. The inability to separate the presence of symptoms from achieving significant immunogenicity in these aroA aroD or aroA aroD htrA strains precludes their use as single dose typhoid vaccines in the formulations tested. Multiple doses of these strains at a lower, safe level may be effective as vectors for foreign antigens.

Characterization of a subunit structure and stability of the recombinant porin from Neisseria gonorrhoeae.

An outer membrane PIA protein from Neisseria gonorrhoeae strain FA19 was expressed in Escherichia coli and refolded in vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50 degrees C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80 degrees C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind with Kd=80 and 130 microM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.

Determination of the unit size of the corneal endothelial cell mosaic from Fourier component image analysis.

Scanning electron micrographs were taken of the central region of the corneal endothelium of cows (Holstein). The cells were outlined and an image overlay generated of approximately 100 cells. Via a Windows-based scanning system, the overlay was subjected to a two-dimensional Fourier transform on a Unix-based system. A custom algorithm was developed (IRIS) to sequentially analyse the Fourier transform pattern. The transform and resultant harmonics spectrum were compared to those obtained from artificial cell mosaics generated from uniform sized symmetrical hexagons. The position, width, and height of the 1st harmonic component of the frequency distribution appears to be derived from average cell-cell border distances across the image. The radial position of the 1st harmonic is inversely related to the unit cell size in the mosaic, i.e. the dominant cell size. The application of such techniques to cell mosaic analyses is discussed.

A program for analyzing the morphological organization of tessellated cellular mosaics.

We have constructed a computer program that allows sequential sets of data from tessellated cellular mosaics to be evaluated such that the presence and position of abnormal cells in the mosaic can be easily, quickly, and quantitatively identified. Conventional parametric analysis of the global morphological organization of large tessellated cellular mosaics does not provide the necessary granularity of analysis to allow for rapid identification of such atypical cellular structures. Our program, written in C using structured programming techniques, provides the needed analytical granularity for detailed studies of subsections of large cellular mosaics, while also providing conventional statistical evaluation of the data set. The architecture of the program is modular and designed as a set of procedures and subprocessess that can either analyze the entire mosaic or discrete sets of cells within the larger sample. The types of morphological heterogeneity (or dishomogeneity) that the program can investigate occur in a variety of biological mosaics where there is a continuum of the structural features, including fairly regular linear stacked arrays of cells or tissue partitions, to less regular mosaics of epithelial or endothelial cell layers, to heterogeneous patterns found on tissue surfaces, such as fluorescein dye-highlighted features on the human conjunctiva. The program has been used to successfully evaluate several different types of mosaics.

Impact of using weighted versus unweighted measures to assess optometry faculty productivity in research.

BACKGROUND: Activities in scholarship and research are commonly self-reported in terms of the number of publications per calendar year or grant-tenure period; individuals might show widely different productivities at different times but specific data on this do not appear to be available for optometry school faculty. METHODS: Using a data base generated from a 20-year activity period at an optometry school, the impact of calculating publication rate by self-reporting or weighted scaling (linear or exponential) according to co-authorship on such publications was assessed for each individual faculty member. RESULTS: Results are presented from a faculty productivity perspective on a calendar year basis or with the faculty members matched for seniority on a year-by-year basis. CONCLUSIONS: The use of weighted scaling to productivity assessment substantially reduced the net output from the faculty in most of the 20 years assessed. The average weighted productivity was generally close to two published articles (excluding book chapters) per year, per faculty member compared to close to three articles per year per faculty member, using self-reported unweighted data.

Using cluster analysis for medical resource decision making.

Escalating costs of health care delivery have in the recent past often made the health care industry investigate, adapt, and apply those management techniques relating to budgeting, resource control, and forecasting that have long been used in the manufacturing sector. A strategy that has contributed much in this direction is the definition and classification of a hospital's output into "products" or groups of patients that impose similar resource or cost demands on the hospital. Existing classification schemes have frequently employed cluster analysis in generating these groupings. Unfortunately, the myriad articles and books on clustering and classification contain few formalized selection methodologies for choosing a technique for solving a particular problem, hence they often leave the novice investigator at a loss. This paper reviews the literature on clustering, particularly as it has been applied in the medical resource-utilization domain, addresses the critical choices facing an investigator in the medical field using cluster analysis, and offers suggestions (using the example of clustering low-vision patients) for how such choices can be made.

Identification of topographical variations in the sizes of cells in a monolayer--application to corneal endothelium.

The cells in the corneal endothelium, do not have a constant geometric arrangement but rather can be organized into widely different patterns or arrays. Conventional statistical analyses of variance in cell size can be used to report that the endothelial mosaic is homogeneous (homomegethous) or heterogeneous (polymegethous) but do not identify the source of the homogeneity or its loss. We present methods that allow for both identification and graphical presentation of regional homogeneity or heterogeneity in the endothelial cell monolayer. Single electron micrographs (containing 180 cells) were taken at the central region of the rabbit corneal endothelium and cell areas measured by planimetry. Special computer programs allow progressive evaluation of variance in all cell areas across the set of cells. Several examples are presented in detail to illustrate different degrees and types of variance for sets of endothelial cells that have small to extremely large ranges of areas (i.e. coefficient of variation values ranging from 0.12 to over 1.0). There also appears to be a mathematically predictable relationship between cell surface area and the number of cell sides. The methods developed should be applicable to quantitative comparisons of cells in monolayers or tissue sections.

Clinical low vision resource usage prediction.

In an era of increased demands and constrained budgets, it is necessary to make the best use of all available resources. This is difficult when specialized vision care, such as low vision clinical assessment, is involved because of the heterogeneity of the patient populations seen by such clinics. PURPOSE. This research attempts to discover if these diverse patient populations can be identified and clustered into groups based upon similarity of clinical resources use. Specifically, the inquiry examines the potential for a low vision patient resource utilization classification scheme at the Low Vision Clinic (LVC) in the Centre for Sight Enhancement (CSE), University of Waterloo. METHODS. From a sample of 99 patients consulting the LVC in a 3-month period, retrospective data collection involved abstracting and coding medical records containing information detailing each patient's demographic, diagnostic, therapeutic, and resource utilization characteristics. Cluster analysis using Hartigan's block clustering algorithm was then applied to the data. A replication study was completed using a sample of 99 patients visiting the LVC 1 year later. RESULTS. Patients can be classified into five iso-resource groups, hereby termed low vision patient resource groups (LVPRGs). The clusters represent a resource consistent and clinically coherent scheme for classifying low vision patients based upon resource requirements. As a measure of repeatability, the groups reemerged in the replication study. CONCLUSIONS. If the groupings demonstrate robustness in a field test, clustering algorithms in general, and LVPRGs in specific, may offer useful tools to enhance resource utilization in the LVC setting.

Human papillomavirus type 1 produces redundant as well as polycistronic mRNAs in plantar warts.

Human papillomavirus type 1 (HPV-1) causes plantar warts. On the basis of previously mapped mRNAs and sequence homologies of HPV-1 to other papillomaviruses, we designed oligonucleotide primers and employed the polymerase chain reaction to recover HPV-1 cDNAs from plantar warts. Seven spliced RNA species were characterized, including three not previously detected, and the coding potentials of each were deduced. The most abundant viral mRNA encodes an E1i--E4 protein. One new species is predicted to encode the full-length E2 protein, and another can, theoretically, encode the E2-C or E1-M proteins, three products that regulate mRNA transcription and DNA replication. One RNA species originating from a novel HPV promoter in the upstream regulatory region has the potential to encode the minor capsid protein L2. A newly recognized E5a open reading frame (ORF) is contained in all mRNAs that are polyadenylated at the E-region poly(A) site and also in a putative L2 mRNA. Three distinct species, two of which are derived from the upstream regulatory region promoter, have the potential to encode the L1 protein; the third species also contains the entire coding region of the E1i--E4 protein 5' to the L1 ORF. Both the E1i--E4 mRNA and the potentially bicistronic L1 mRNA are derived from a promoter located in the E7 ORF. We uncovered no evidence of alternatively spliced mRNAs that could account for the multiple, abundant E4 proteins in plantar warts, suggesting that posttranslational modification is mainly responsible for the observed protein heterogeneity.

Distribution of gonococcal lipopolysaccharide biosynthesis genes among strains of Neisseria gonorrhoeae and other neisserial species.

A plasmid, pTME6, containing Neisseria gonorrhoeae lipopolysaccharide biosynthesis genes was used as a probe to analyze DNA from strains of N. gonorrhoeae, N. meningitidis and various commensal Neisseria by Southern blotting. Chromosomal DNA from 26 gonococcal strains probed with 32P-labeled pTME6 produced five different hybridization patterns. No correlation between hybridization pattern and auxotype, serotype, serum sensitivity or SDS-urea-PAGE migration of LPS was observed. DNA from strains of N. meningitidis, N. lactamica and N. cinerea, but not other commensal Neisseria species, hybridized strongly to pTME6.

Unions and health care cost inflation.

Since the 1974 Taft-Hartley amendments gave nonproprietary hospital employees the right to organize and bargain collectively, the rate of growth of work stoppages has decelerated and the number of man days lost has been reduced. Wage rates, earnings differential, and fringe benefits have not been significantly improved by unionization. And little evidence supports the contention that unionization has contributed substantially to the inflation experienced in the health care industry.