Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

MR image-based measurement of rates of change in volumes of brain structures. Part 1: Method and validation

A detailed analysis procedure is described for evaluating rates of volumetric change in brain structures based on structural magnetic resonance (MR) images. In this procedure, a series of image processing tools have been employed to address the problems encountered in measuring rates of change based on structural MR images. These tools include an algorithm for intensity non-uniforniity correction, a robust algorithm for three-dimensional image registration with sub-voxel precision and an algorithm for brain tissue segmentation. However, a unique feature in the procedure is the use of a fractional volume model that has been developed to provide a quantitative measure for the partial volume effect. With this model, the fractional constituent tissue volumes are evaluated for voxels at the tissue boundary that manifest partial volume effect, thus allowing tissue boundaries be defined at a sub-voxel level and in an automated fashion. Validation studies are presented on key algorithms including segmentation and registration. An overall assessment of the method is provided through the evaluation of the rates of brain atrophy in a group of normal elderly subjects for which the rate of brain atrophy due to normal aging is predictably small. An application of the method is given in Part 11 where the rates of brain atrophy in various brain regions are studied in relation to normal aging and Alzheimer's disease. (C) 2002 Elsevier Science Inc. All rights reserved.

Knowledge of the national emergency telephone number and prevalence and characteristics of those trained in CPR in Queensland: baseline information for targeted training interventions

Members of the community contribute to survival from out-of-hospital cardiac arrest by contacting emergency medical services and performing cardiopulmonary resuscitation (CPR) prior to the arrival of an ambulance. In Australia there is a paucity of information of the extent that community members know the emergency telephone number and are trained in CPR. A survey of Queensland adults (n = 4490) was conducted to ascertain current knowledge and training levels and to target CPR training. Although most respondents (88.3%) could state the Australian emergency telephone number correctly, significant age differences were apparent (P < 0.001). One in five respondents aged 60 years and older could not state the emergency number correctly. While just over half the respondents (53.9%) had completed some form of CPR training, only 12.1% had recent training. Older people were more likely to have never had CPR training than young adults. Additional demographic and socio-economic differences were found between those never trained in CPR and those who were. The results emphasise the need to increase CPR training in those aged 40 and over, particularly females, and to increase the awareness of the emergency telephone number amongst older people. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Cuticular hydrocarbons of Drosophila birchii and D-serrata: Identification and role in mate choice in D-serrata

The cuticular hydrocarbon compositions of two sympatric species of Australian Drosophila in the montium subgroup of the melanogaster group that use cuticular hydrocarbons in mate recognition have been characterized. Drosophila birchii has 34 components in greater than trace amounts, with a carbon number range of C-20 to C-33. Drosophila serrata has 21 components above trace level and a carbon number range of C-24 to C-31. These two species share eight hydrocarbon components, with all but two of them being monoenes. For both species, the (Z)-9-monoenes are the predominant positional isomer. The hydrocarbons of D. birchii are n-alkanes, n-alkenes (Z)-5-, (Z)-7-, (Z)-9-, and (Z)-11-), low to trace levels of homologous (Z,Z)-7,11- and (Z,Z)-9,13-dienes; and trace amounts of (Z,Z)-5,9- C-25:2, a major component of D. serrata. Only one methyl branched hydrocarbon was detected (2-methyl C-28), and it occurred at very low levels. The hydrocarbons of D. serrata are dominated by a homologous series of (Z,Z)-5,9-dienes, and notably, are characterized by the apparent absence of n-alkanes. Homologous series of (Z)-5-, (Z)-7-, and (Z)-9- alkenes are also present in D. serrata as well as 2-methyl alkanes. Drosophila serrata females display strong directional mate choice based on male cuticular hydrocarbons and prefer D. serrata males with higher relative abundances of the 2-methyl alkanes, but lower relative abundances of (Z,Z)-5,9- C-24:2 and (Z)-9-C-25:1.

Identification of the enamelin (g.8344delG) mutation in a new kindred and presentation of a standardized ENAM nomenclature

The amelogenesis imperfectas (Al) area geneticatly heterogeneous group of diseases that result in defective development of tooth enamel. Although X-linked, autosomal. dominant and autosomal. recessive forms of Al have been clinically characterized, only two genes (AMELX and ENAM) have been associated with Al. To date, three enamelin (ENAM) mutations have been identified. These mutations cause phenotypically diverse forms of autosomal. dominant Al. Detailed phenotype-genotype correlations have not been performed for autosomal. dominant Al due to ENAM mutations. We identified a previously unreported kindred segregating for the ENAM mutation, g.8344delG. Light and electron microscopy analyses of unerupted permanent teeth show the enamel is markedly reduced in thickness, Lacks a prismatic structure and has a laminated appearance. Taken together these histological features support the enamelin protein as being critical for the development of a normal. enamel. thickness and that it Likely has a role in regulating c-axis crystallite growth. Because there is growing molecular and phenotypic diversity in the enamelin defects, it is critical to have a nomenclature and numbering system for characterizing these conditions. We present a standardized nomenclature for ENAM mutations that will allow consistent reporting and communication. (C) 2003 Elsevier Science Ltd. All rights reserved.

Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: Biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Quality of life assessment in the community-dwelling elderly: Validation of the Assessment of Quality of Life (AQoL) Instrument and comparison with the SF-36

Measurement of Health-Related Quality of Life (HRQoL) of the elderly requires instruments with demonstrated sensitivity, reliability, and validity, particularly with the increasing proportion of older people entering the health care system. This article reports the psychometric properties of the 12-item Assessment of Quality of Life (AQoL) instrument in chronically ill community-dwelling elderly people with an 18-month follow-up. Comparator instruments included the SF-36 and the OARS. Construct validity of the AQoL was strong when examined via factor analysis and convergent and divergent validity against other scales. Receiver Operator Characteristic (ROC) curve analyses and relative efficiency estimates indicated the AQoL is sensitive, responsive, and had the strongest predicative validity for nursing home entry. It was also sensitive to economic prediction over the follow-up. Given these robust psychometric properties and the brevity of the scale, AQoL appears to be a suitable instrument for epidemiologic studies where HRQoL and utility data are required from elderly populations. (C) 2003 Elsevier Science Inc. All rights reserved.

The Knowledge and Use of the Balanced Scorecard in Portugal: Empirical Evidence of the 250 Largest Companies

Balanced Scorecard (BSC) is recognized, both in the academic and business world, as one of the most powerful strategic management accounting tools. Thus, we launched in October 2004 a questionnaire survey applied to the 250 largest Portuguese companies aiming at observing the knowledge, use, and companies’ characteristics which are adopting this management instrument. Despite the majority of the companies inquired recognize BSC more as a strategic management tool than a performance valuation system, the results show that there is still a reduced and recent utilization of BSC in Portugal. Similarly to other countries Portugal is still in the initial state of BSC utilization. Our work has shown that the companies that use more BSC belong mainly to the secondary sector of industry. Nevertheless, unlike other studies, we did not get empirical evidence on the influence of variables such as geographical localization, dimension and internationalization, in the use and knowledge of BSC in Portugal.

Validation of a measurement scale for the relationship between the orientation to corporate social responsibility and other business strategic variables

The importance of Social Responsibility (SR) is higher if this business variable is related with other ones of strategic nature in business activity (competitive success that the company achieved, performance that the firms develop and innovations that they carries out). The hypothesis is that organizations that focus on SR are those who get higher outputs and innovate more, achieving greater competitive success. A scale for measuring the orientation to SR has defined in order to determine the degree of relationship between above elements. This instrument is original because previous scales do not exist in the literature which could measure, on the one hand, the three classics sub-constructs theoretically accepted that SR is made up and, on the other hand, the relationship between SR and the other variables. As a result of causal relationships analysis we conclude with a scale of 21 indicators, validated scale with a sample of firms belonging to the Autonomous Community of Extremadura and it is the first empirical validation of these dimensions we know so far, in this context.

Virtual communities of practice: investigating motivations and constraints in the processes of knowledge creation and transfer

With accelerated market volatility, faster response times and increased globalization, business environments are going through a major transformation and firms have intensified their search for strategies which can give them competitive advantage. This requires that companies continuously innovate, to think of new ideas that can be transformed or implemented as products, processes or services, generating value for the firm. Innovative solutions and processes are usually developed by a group of people, working together. A grouping of people that share and create new knowledge can be considered as a Community of Practice (CoP). CoP’s are places which provide a sound basis for organizational learning and encourage knowledge creation and acquisition. Virtual Communities of Practice (VCoP's) can perform a central role in promoting communication and collaboration between members who are dispersed in both time and space. Nevertheless, it is known that not all CoP's and VCoP's share the same levels of performance or produce the same results. This means that there are factors that enable or constrain the process of knowledge creation. With this in mind, we developed a case study in order to identify both the motivations and the constraints that members of an organization experience when taking part in the knowledge creating processes of VCoP's. Results show that organizational culture and professional and personal development play an important role in these processes. No interviewee referred to direct financial rewards as a motivation factor for participation in VCoPs. Most identified the difficulty in aligning objectives established by the management with justification for the time spent in the VCoP. The interviewees also said that technology is not a constraint.

Monte Carlo modeling and simulations of the High Definition (HD120) micro MLC and validation against measurements for a 6 MV beam

Purpose: The most recent Varian® micro multileaf collimator(MLC), the High Definition (HD120) MLC, was modeled using the BEAMNRCMonte Carlo code. This model was incorporated into a Varian medical linear accelerator, for a 6 MV beam, in static and dynamic mode. The model was validated by comparing simulated profiles with measurements. Methods: The Varian® Trilogy® (2300C/D) accelerator model was accurately implemented using the state-of-the-art Monte Carlo simulation program BEAMNRC and validated against off-axis and depth dose profiles measured using ionization chambers, by adjusting the energy and the full width at half maximum (FWHM) of the initial electron beam. The HD120 MLC was modeled by developing a new BEAMNRC component module (CM), designated HDMLC, adapting the available DYNVMLC CM and incorporating the specific characteristics of this new micro MLC. The leaf dimensions were provided by the manufacturer. The geometry was visualized by tracing particles through the CM and recording their position when a leaf boundary is crossed. The leaf material density and abutting air gap between leaves were adjusted in order to obtain a good agreement between the simulated leakage profiles and EBT2 film measurements performed in a solid water phantom. To validate the HDMLC implementation, additional MLC static patterns were also simulated and compared to additional measurements. Furthermore, the ability to simulate dynamic MLC fields was implemented in the HDMLC CM. The simulation results of these fields were compared with EBT2 film measurements performed in a solid water phantom. Results: Overall, the discrepancies, with and without MLC, between the opened field simulations and the measurements using ionization chambers in a water phantom, for the off-axis profiles are below 2% and in depth-dose profiles are below 2% after the maximum dose depth and below 4% in the build-up region. On the conditions of these simulations, this tungsten-based MLC has a density of 18.7 g cm− 3 and an overall leakage of about 1.1 ± 0.03%. The discrepancies between the film measured and simulated closed and blocked fields are below 2% and 8%, respectively. Other measurements were performed for alternated leaf patterns and the agreement is satisfactory (to within 4%). The dynamic mode for this MLC was implemented and the discrepancies between film measurements and simulations are within 4%. Conclusions: The Varian® Trilogy® (2300 C/D) linear accelerator including the HD120 MLC was successfully modeled and simulated using the Monte CarloBEAMNRC code by developing an independent CM, the HDMLC CM, either in static and dynamic modes.

Knowledge, attitude and practice of mammography among women users of public health services

OBJECTIVE: To evaluate knowledge, attitude and practice related to mammography among women users of local health services, identifying barriers to its performance. METHODS: A total of 663 women were interviewed at 13 local health centers in a city of Southeastern Brazil, in 2001. Interviewees were randomly selected at each center and they were representative from different socioeconomic conditions. The number of interviewees at each center was proportional to monthly mean appointments. For data analysis, answers were described as knowledge, attitude, practice and their respective adequacies and then they were correlated with control variables through the chi-square test. RESULTS: Only 7.4% of the interviewees had adequate knowledge on mammography, while 97.1% of women had an adequate attitude. The same was seen for the practice of mammography that was adequate in 35.7% of the cases. The main barrier to mammography was lack of referral by physicians working at the health center (81.8%). There was an association between adequacy of attitude and five years or more of education and being married. There was also an association between adequacy of mammography practice and being employed and family income up to four minimum wages. CONCLUSIONS: Women users of local health services had no adequate knowledge and practice related to mammography despite having an adequate attitude about this exam.

Validation of a single-extraction procedure for sequential analysis of vitamin E, cholesterol, fatty acids, and total fat in seafood

Food lipid major components are usually analyzed by individual methodologies using diverse extractive procedures for each class. A simple and fast extractive procedure was devised for the sequential analysis of vitamin E, cholesterol, fatty acids, and total fat estimation in seafood, reducing analyses time and organic solvent consumption. Several liquid/liquid-based extractive methodologies using chlorinated and non-chlorinated organic solvents were tested. The extract obtained is used for vitamin E quantification (normal-phase HPLC with fluorescence detection), total cholesterol (normal-phase HPLC with UV detection), fatty acid profile, and total fat estimation (GC-FID), all accomplished in <40 min. The final methodology presents an adequate linearity range and sensitivity for tocopherol and cholesterol, with intra- and inter-day precisions (RSD) from 3 to 11 % for all the components. The developed methodology was applied to diverse seafood samples with positive outcomes, making it a very attractive technique for routine analyses in standard equipped laboratories in the food quality control field.