The New Zealand priority criteria project. Part 1: Overview.

New Zealand restructured its health system in 1992 with the aim of achieving greater levels of assessment and accountability in the publicly funded health sector. A committee was established specifically to advise the minister of health on the kinds, and relative priorities, of health services that should be publicly funded. One of its projects has been to develop standardised sets of criteria to assess the extent of benefit expected from elective surgical procedures. These have been developed with the help of professional advisory groups using a modified Delphi technique to reach consensus. So far the committee has developed criteria for cataract surgery, coronary artery bypass grafting, hip and knee replacement, cholecystectomy, and tympanostomy tubes for otitis media with effusion. These criteria incorporate both clinical and social factors. Use of priority criteria to ensure consistency and transparency regarding patients'' priority for surgery is required for access to a dedicated NZ$130m (pounds 57m; US$90m) pool of money, created to help eliminate surgical waiting lists and move to booking systems. The criteria will also be used in surgical outcome studies, currently in the planning phase.     

Model of oxygen transport limitations in hollow fiber bioreactors

Axial and radial oxygen depletion are believed to be critical scale-limiting factors in the design of cell culture hollow fiber bioreactors. A mathematical analysis of oxygen depletion has been performed in order to develop effectiveness factor plots to aid in the scaling of hollow fiber bioreactors with cells immobilized in the shell-side. Considerations of the lumen mass transport resistances and the axial gradients were added to previous analyses of this immobilization geometry. An order of magnitude analysis was used to evaluate the impact of the shell-side convective fluxes on the oxygen transport. A modified Thiele modulus and a lumen and membrane resistance factor have been derived from the model. Use of these terms in the effectiveness factor plots results in a considerable simplification of the presentation and use of the model. Design criteria such as fiber dimensions and spacing, reactor lengths, and recycle flow rates can be selected using these plots. Model predictions of the oxygen limitations were compared to experimental measurements of the axial cell distributions in a severely oxygen limited hollow fiber bioreactor. Despite considerable uncertainty in our parameters and nonidealities in hollow fiber geometry, the cell distribution correlated well with the modeling results.     

Improved Microscopic Detection of Bacteriuria

A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as “significant” by the conventional laboratory culture. More importantly, it immediately reveals the presence of bacteria, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.     

Analyse des criteres de choix des materiels et technologies a l'hopital

Decision-making in hospital, when a biomedical instrumentation investment has to be decided, is not simple because of the large amount of criteria which should be considered. To show the most important criteria in decision-making, their respective weight and their interdependencies, we used multi-dimensional analysis methods. A quasi-exhaustive list of criteria has been constructed and sent to all the heads of medical departments, the directors and the clinical engineers of the French university hospitals, in order to obtain their appreciation of the influence of these criteria in their own decision-making experience. The application of factorial analysis methods to the answers gives two main results. There is no characteristic behaviour of professional groups, such as engineers, directors, surgeons, radiologist, etc.… A list of 21 criteria, which are the most influential in decision-making, can be identified. This result will be emphasized in order to develop a tool which allows one to measure the adequacy of a biomedical instrument that shows a clinical need.     

Marrow grown in long-term culture can be used for autologous transplantation in the treatment of acute myeloid leukaemia

Leukaemic cells can be selectively depleted in the conditions of long-term bone marrow culture (LTBMC). Use can be made of LTBMC to purge bone marrow of leukaemic cells in autologous bone marrow transplantation (ABMT).     

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.     

Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins

Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B. If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.     

On-line analysis of avermectin fermentation cell growth kinetics in an industrial pilot plant

Experimental data are presented which show that on-line calculation of oxygen uptake rate can be used to estimate the cell concentration of Streptomyces avermitilis during the active growth phase of this fermentation. Moreover, by dividing the oxygen uptake rate by the total oxygen consumed, an on-line estimate of specific growth rate of this culture can be generated. A theoretical basis is provided for this model. Use of a mass spectrometer for vent gas analysis coupled with computer data acquisition has made this information both very accurate and readily available. Examples are given which illustrate the kinetics of the avermectin fermentation as well as the effect of a temperature shift on the specific growth rate.     

Adipocyte studies: systems for investigating effects of growth hormone and other chronically acting hormones

Adipose tissue is very amenable to study in vitro. Collagenase digestion yields free adipocytes which usually respond well to acute stimulation/inhibition by hormones and other factors. Chronic effects of hormones are best studied using explants of adipose tissue which, from some species (e.g. sheep), can be maintained in culture for up to a week without loss of function. Alternatively, preadipocytes can be readily isolated from adipose tissue and induced to proliferate and differentiate in culture, while various adipocyte-like cell-lines have been established, which can be used for chronic studies. Use of these various systems for investigating the mechanisms of action of growth hormone are described.     

5-Aminolevulinic Acid: Production by Fermentation,and Agricultural and Biomedical Applications

Abstract

Research involving differentiated embryonic stem (ES) cells may revolutionize the study of liver disease, improve the drug discovery process, and assist in the development of stem-cell-based clinical therapies. Generation of ES cell-derived hepatic tissue has benefited from an understanding of the cytokines, growth factors and biochemical compounds that are essential in liver development, and this knowledge has been used to mimic some aspects of embryonic development in vitro. Although great progress has been made in differentiating human ES cells into liver cells, current protocols have not yet produced cells with the phenotype of a mature hepatocyte. There is a of disease models have been examined concerning whether stem cells can correct liver disease. It is a bit premature to conclude that hepatocytes can be generated from non-hepatic cells in culture that will be clinically useful. Standard criteria will need to be developed to assess the extent to which human stem cell-derived hepatocytes have been produced.     

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

Background

Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods

A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results

The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions

Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.     

Recommended protocol for the BALB/c 3T3 cell transformation assay

The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.     

Scoring criteria of aneuploidy frequency in interphase nuclei by fish-analysis

Principles and approaches of assessment of numerical chromosome abnormalities in interphase nuclei by fluorescent in situ hybridization technology are reviewed. The authors' own scoring criteria of results of hybridization of centromere-specific DNA-probes with chromosomal targets in somatic cells by dual color FISH-analysis are suggested. Use of these scoring criteria allows to reduce the level of artificial hypoploidy, whose its frequency becomes similar to that of hyperploidy. This fact indicates that chromosomal nondisjunction is a major mechanism of aneuploidy induction, rather than of chromosome loss.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

ESTprep: preprocessing cDNA sequence reads

MOTIVATION: High accuracy of data always governs the large-scale gene discovery projects. The data should not only be trustworthy but should be correctly annotated for various features it contains. Sequence errors are inherent in single-pass sequences such as ESTs obtained from automated sequencing. These errors further complicate the automated identification of EST-related sequencing. A tool is required to prepare the data prior to advanced annotation processing and submission to public databases. RESULTS: This paper describes ESTprep, a program designed to preprocess expressed sequence tag (EST) sequences. It identifies the location of features present in ESTs and allows the sequence to pass only if it meets various quality criteria. Use of ESTprep has resulted in substantial improvement in accurate EST feature identification and fidelity of results submitted to GenBank. AVAILABILITY: The program is freely available for download from http://genome.uiowa.edu/pubsoft/software.html     

Studies on Mineral Nutrient Uptake using Tissue Culture Derived Plants of Banana (Musa sp)

Banana (Musa sp) plants regenerated from in vitro culture were employed to study varietal differences of ³²P uptake. The results demonstrated that the selected varieties exhibited considerable variation for absorption, translocation and transport index of PO₄ uptake. Use of tissue cultured derived plants can be efficiently utilised to analyse varietal differences in ³²P uptake.     

MALDI-TOF-Based Dermatophyte Identification

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100 % for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.     

Growth of cultured mammalian cells on secondary glucose sources

Mammalian cells can grow in culture at very low glucose concentrations. They can also grow using starch or maltose as secondary sources of glucose if hydrolytic enzymes (amylase and/or maltase) are available to release the glucose. The serum supplement in the culture medium provides these enzymes in amount adequate to permit growth at as rapid a rate as when free glucose is added. Owing to the relatively slow liberation of glucose from the secondary sources, the cells produce less lactic acid, and the culture medium does not become acidic.If the amount of hydrolytic enzyme in the serum supplement is reduced by heat inactivation, the rate of glucose liberation is further reduced. As a result, glucose continues to be released into the medium even at high cell densities, when all glucose added directly to control cultures has been consumed at a time. For this reason, the cells survive longer at high density on secondary glucose sources than on free glucose. Use of such a culture system should have important practical advantages in maintaining dense cultures of any mammalian cell type.Medium containing secondary glucose sources and serum whose hydrolytic enzymes have been completely inactivated should be a selective medium for the corresponding cellular enzymes. Attempts to select for cell lines able to grow using their own amylase or maltase were not successful, but calculations based on embryonic pancreatic cells, known to synthesize amylase, showed that the amount of enzyme required should be quite low in comparison with that present in the differentiated state. The possibilities of selection for a differentiated function in cell culture have been very little explored, and such an approach may be fruitful if applied to the right cell types.     

Analysis of a simulated microarray dataset: Comparison of methods for data normalisation and detection of differential expression (Open Access publication)

Microarrays allow researchers to measure the expression of thousands of genes in a single experiment. Before statistical comparisons can be made, the data must be assessed for quality and normalisation procedures must be applied, of which many have been proposed. Methods of comparing the normalised data are also abundant, and no clear consensus has yet been reached. The purpose of this paper was to compare those methods used by the EADGENE network on a very noisy simulated data set. With the a priori knowledge of which genes are differentially expressed, it is possible to compare the success of each approach quantitatively. Use of an intensity-dependent normalisation procedure was common, as was correction for multiple testing. Most variety in performance resulted from differing approaches to data quality and the use of different statistical tests. Very few of the methods used any kind of background correction. A number of approaches achieved a success rate of 95% or above, with relatively small numbers of false positives and negatives. Applying stringent spot selection criteria and elimination of data did not improve the false positive rate and greatly increased the false negative rate. However, most approaches performed well, and it is encouraging that widely available techniques can achieve such good results on a very noisy data set.