Comparative study of thermal stability of magnetostrictive biosensor between two kinds of biorecognition elements.

Magnetostrictive biosensors specific to Salmonella typhimurium were prepared by immobilizing antibody or phage as biorecognition elements onto the magnetostrictive sensor platform. The sensors were stored at temperatures of 25 °C (room temperature), 45 °C and 65 °C, respectively, and the ability to bind S. typhimurium was detected by testing the resonant frequency shift using a HP network analyzer after exposure to 1 mL of 1×10(9) cfu/mL of S. typhimurium at a predetermined schedule. The binding of S. typhimurium to biosensors was confirmed by Scanning Electron Microscopy (SEM). The results showed that there existed an initial sudden drop in the average density of S. typhimurium bound to the biosensor surface versus duration at different temperatures for the two kinds of recognition elements, and the binding ability to S. typhimurium of phage-immobilized biosensors was much better than that of antibody-immobilized biosensors, with longevity longer than 30 days at all tested temperatures, though decreasing gradually over the testing period. While the longevity of antibody-immobilized biosensors was only about 30, 8 and 5 days at room temperature (25 °C), 45 °C and 65 °C, respectively. Meanwhile, the activation energy of the two kinds of biosensors was investigated, and it was found that phage immobilized sensors showed much higher activation energy than antibody immobilized sensors, which resulted in less dependency on temperature and thus having much better thermal stability than antibody immobilized sensors.

Metabolic crisis after traumatic brain injury is associated with a novel microdialysis proteome.

BACKGROUND: To examine if the metabolic distress after traumatic brain injury (TBI) is associated with a unique proteome. METHODS: Patients with severe TBI prospectively underwent cerebral microdialysis for the initial 96 h after injury. Hourly sampling of metabolism was performed and patients were categorized as having normal or abnormal metabolism as evidenced by the lactate/pyruvate ratio (LPR) threshold of 40. The microdialysate was frozen for proteomic batch processing retrospectively. We employed two different routes of proteomic techniques utilizing mass spectrometry (MS) and categorized as diagnostic and biomarker identification approaches. The diagnostic approach was aimed at finding a signature of MS peaks which can differentiate these two groups. We did this by enriching for intact peptides followed by MALDI-MS analysis. For the biomarker identification approach, we applied classical bottom-up (trypsin digestion followed by LC-MS/MS) proteomic methodologies. RESULTS: Five patients were studied, 3 of whom had abnormal metabolism and 2 who had normal metabolism. By comparison, the abnormal group had higher LPR (1609 +/- 3691 vs. 15.5 +/- 6.8, P < 0.001), higher glutamate (157 +/- 84 vs. 1.8 +/- 1.4 microM, P < 0.001), and lower glucose (0.27 +/- 0.35 vs. 1.8 +/- 1.1 mmol/l, P < 0.001). The abnormal group demonstrated 13 unique proteins as compared with the normal group in the microdialysate. These proteins consisted of cytoarchitectural proteins, as well as blood breakdown proteins, and a few mitochondrial proteins. A unique as yet to be characterized peptide was found at m/z (mass/charge) 4733.5, which may represent a novel biomarker of metabolic distress. CONCLUSION: Metabolic distress after TBI is associated with a differential proteome that indicates cellular destruction during the acute phase of illness. This suggests that metabolic distress has immediate cellular consequences after TBI.

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors.

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.

The effect of salt and phage concentrations on the binding sensitivity of magnetoelastic biosensors for Bacillus anthracis detection.

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.

Diabetic fibrous mastopathy.

Diabetic fibrous mastopathy is reported in a 37-year-old premenopausal woman. A known case of insulin-dependent diabetes mellitus, she presented with bilateral hard breast lumps, which were suggestive of malignancy on both ultrasonography and mammography. Fine-needle aspiration cytology and core biopsy showed fibrosis. An incisional biopsy further excluded malignancy and was conclusive for diabetic fibrous mastopathy.

Rapid and sensitive magnetoelastic biosensors for the detection of Salmonella typhimurium in a mixed microbial population.

In this article, we report the results of an investigation into the performance of a wireless, magnetoelastic biosensor designed to selectively detect Salmonella typhimurium in a mixed microbial population. The Langmuir-Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors (2 x 0.4 x 0.015 mm). Bacterial binding to the antibody on the sensor surface changes the resonance parameters, and these changes were quantified as a shift in the sensor's resonance frequency. Response of the sensors to increasing concentrations (5 x 10(1) to 5 x 10(8) cfu/ml) of S. typhimurium in a mixture of extraneous foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was studied. A detection limit of 5 x 10(3) cfu/ml and a sensitivity of 139 Hz/decade were observed for the 2 x 0.4 x 0.015 mm sensors. Binding kinetics studies have shown that the dissociation constant (K(d)) and the binding valencies for water samples spiked with S. typhimurium was 435 cfu/ml and 2.33 respectively. The presence of extraneous microorganisms in the mixture did not produce an appreciable change in the biosensor's dose response behavior.

Effects of high-pressure processing on Listeria monocytogenes, spoilage microflora and multiple compound quality indices in chilled cold-smoked salmon.

AIMS: To evaluate the effect of high-pressure processing (HPP) on Listeria monocytogenes, microbial and chemical changes and shelf-life in chilled cold-smoked salmon (CSS). METHODS AND RESULTS: First, challenge tests with L. monocytogenes were carried out using HPP of the product at 0.1 (control), 150, 200 and 250 MPa. Secondly, storage trials with the naturally contaminated product and HPP at 0.1 (control) and 200 MPa were realized. Shelf-life, microbial changes and chemical changes were determined and existing predictive models and multiple compound quality indices evaluated. HPP with 250 MPa did not inactivate L. monocytogenes but significant lag phases of 17 and 10 days were observed at ca 5 and 10 degrees C, respectively. HPP with 200 MPa had a marked effect on both colour and texture of CSS. CONCLUSIONS: High-pressure processing was unable to prevent growth of L. monocytogenes or spoilage of chilled CSS. Existing mathematical models allowed growth rates of L. monocytogenes and shelf-life of samples without high-pressure treatments to be predicted. SIGNIFICANCE AND IMPACT OF THE STUDY: High-pressure processing seems more appropriate for new types of salmon products than for a classical product like CSS where consumers expect specific quality attributes.

Global methylation profiling of lung cancer identifies novel methylated genes.

Epigenetic changes, including DNA methylation, are a common finding in cancer. In lung cancers methylation of cytosine residues may affect tumor initiation and progression in several ways, including the silencing of tumor suppressor genes through promoter methylation and by providing the targets for adduct formation of polycyclic aromatic hydrocarbons present in combustion products of cigarette smoke. Although the importance of aberrant DNA methylation is well established, the extent of DNA methylation in lung cancers has never been determined. Restriction landmark genomic scanning (RLGS) is a highly reproducible two-dimensional gel electrophoresis that allows the determination of the methylation status of up to 2000 promoter sequences in a single gel. We selected 1184 CpG islands for RLGS analysis and determined their methylation status in 16 primary non-small cell lung cancers. Some tumors did not show methylation whereas others showed up to 5.3% methylation in all CpG islands of the profile. Cloning of 21 methylated loci identified 11 genes and 6 ESTs. We demonstrate that methylation is part of the silencing process of BMP3B in primary tumors and lung cancer cell lines.

DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors.

Histone acetylation has long been associated with transcriptional activation, whereas conversely, deacetylation of histones is associated with gene silencing and transcriptional repression. Here we report that inhibitors of histone deacetylase (HDAC), depsipeptide and trichostatin A, induce apoptotic cell death in human lung cancer cells as demonstrated by DNA flow cytometry and Western immunoblot to detect cleavage of poly(ADP-ribose) polymerase. This HDAC inhibitorinduced apoptosis is greatly enhanced in the presence of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The HDAC inhibitor-induced apoptosis appears to be p53 independent, because no change in apoptotic cell death was observed in H1299 cells that expressed exogenous wild-type p53 (H1299 cells express no endogenous p53 protein). To further investigate the mechanism of DAC-enhanced, HDAC inhibitor-induced apoptosis, we analyzed histone H3 and H4 acetylation by Western immunoblotting. Results showed that depsipeptide induced a dose-dependent acetylation of histones H3 and H4, which was greatly increased in DAC-pretreated cells. By analyzing the acetylation of specific lysine residues at the amino terminus of histone H4 (Ac-5, Ac-8, Ac-12, and Ac-16), we found that the enhancement of HDAC inhibitor-induced acetylation of histones in the DAC-pretreated cells was not lysine site specific. These results demonstrate that DNA methylation status is an important determinant of apoptotic susceptibility to HDAC inhibitors.

Benchmarking graduate health administration programs: stakeholders' perceptions.

The purpose of this study was to identify and compare among key stakeholders the factors in graduate health care administration education that are perceived to be important for ranking, or benchmarking, based on the opinions of those stakeholders, i.e., program directors, faculty, graduate students, and accrediting agency commissioners. We used an original survey to obtain stakeholders' perceptions and opinions about important process and outcome measures. We sent it to all ACEHSA-accredited graduate health care administration programs in the United States, Canada, and Puerto Rico; to full-time faculty members in each program; to three current graduate students in each program, and to all ACEHSA commissioners. We performed frequency of responses, Analysis of Variances (ANOVA) tests, and Dunnett T3 tests. A response rate of 32 percent (n = 156) was achieved for all stakeholders. A total of 67 percent of all respondents reported that benchmarking graduate health care administration programs was important. The study results revealed a significant difference between populations on the importance of evaluating certain process and outcome measures related to curriculum, research, student characteristics, and resources. However, most of the stakeholders reported that curriculum, faculty, and graduate student performance were the key quality indicators of a program. The results of this study provide preliminary information for health care administration programs to begin to develop an educational benchmarking effort.

Age-related defects in Th1 and Th2 cytokine production by human T cells can be dissociated from altered frequencies of CD45RA+ and CD45RO+ T cell subsets.

The present study investigated whether age-related changes in the production of Th1 and Th2 cytokines by human T cells might be linked to altered frequencies of naive (CD45RA+) and memory (CD45RO+) T cell subsets. T cells from healthy elderly humans (n = 32) stimulated with anti-CD3epsilon monoclonal antibody OKT3 plus PMA produced significantly lower levels of IL-2 and IFNgamma (Th1 type) and of IL-4 (Th2 type) cytokines compared with T cells from young subjects. Although considerable heterogeneity was observed in the levels of cytokines produced by activated T cells from elderly individuals, linear regression analysis failed to demonstrate any significant shift in Th1 to Th2 type cytokine profiles of human T cells during aging. Sufficient T cells were available from eighteen elderly subjects to quantitate the levels of cytokine production in parallel with flow cytometry analysis of the frequencies of CD45RA+ naive and CD45RO+ memory T cells. Compared with the group of young subjects, the elderly group exhibited significant decreases in the frequencies of naive T cells with reciprocal increases in memory T cells. However, defects in Th1 and Th2 cytokine production were not significantly correlated with altered frequencies of naive/memory T cells among elderly individuals. In addition, those elderly individuals with normal frequencies of naive/memory T cells exhibited decreases in cytokine production comparable to the reductions observed for elderly donors with alterations in the frequencies of naive/memory T cells. These findings suggest that age-related defects in Th1 and Th2 cytokine production cannot be attributed entirely to alterations in the frequencies of naive/memory T cell subsets and point toward intrinsic aberrancies within human T cell cytokine networks during aging.

Downregulation of class II antigen expression by FGF-heparin complexes is due primarily to heparin effect.

Fibroblast growth factors are thought to play a role in the pathogenesis of autoimmune inflammation. The evidence linking these growth factors to autoimmunity stems in part from their presence in mononuclear cells from inflammatory sites during disease processes. We sought to further dissect the mechanisms through which fibroblast growth factors might affect the inflammatory response. Peritoneal macrophages from autoimmune MRL 1pr/1pr mice and congenic wild-type MRL +/+ mice were cultured for 72 hours in the presence of either IFN-gamma, heparin, FGF-1, FGF-2, FGF-1 plus heparin, FGF-2 plus heparin or medium alone. Expression of MHC class II (I-Ak and I-Ek) antigens were analyzed using direct immunofluorescence and flow cytometry. As expected, at baseline there were higher numbers of I-Ak bearing cells in elicited peritoneal cells from 1pr mice relative to +/+ cells (70.8 +/- 14.9 versus 43.4 +/- 19.7, p = 0.046). Expression of I-Ak and I-Ek and percentage of I-E bearing cells were essentially the same between strains. Cells from 1pr and +/+ mice displayed reductions in the percentage of I-Ak expressing cells following culture with FGF-1 plus heparin and FGF-2 plus heparin. Similarly, cells from both 1pr and +/+ mice displayed significant reductions from baseline I-Ak expression following culture with FGF-1 and FGF-2 in the presence of heparin. Similar reductions were seen in cells from both strains cultured with heparin alone. No change from baseline was discernible when cells were cultured in the presence of FGF-1 or FGF-2 alone. Titration studies showed a maximum heparin effect at 5 units/ml culture. However, reduced amounts of heparin in the cell culture were directly proportional to decreased levels of I-Ak expression. These results suggest that cells from autoimmune MRL 1pr/1pr mice and wild type MRL +/+ mice respond similarly with a general reduction of I-Ak expression and a decrease in the percentage of I-Ak bearing cells in response to heparin with little discernible effect from addition of either FGF-1 or FGF-2. This change in class II expression suggests that the heparin-heparan component in FGF-heparin complexes may serve to downregulate class II expression during inflammation.

Phosphorylation and coupling of zeta-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans.

Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of zeta-chains by the regulated activities of protein tyrosine kinases p56(lck) and p59(fyn) as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of zeta-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of zeta-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of zeta-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated zeta-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3epsilon ligated T-cells from young subjects. Analyses of the levels of zeta-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in zeta-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in zeta-chain phosphorylations were not due to diminished frequencies of CD3epsilon+ cells or densities of CD3epsilon surface receptors and could be observed without reductions in epsilon-chain phosphorylations. These results suggest that aberrancies of zeta-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of zeta-chains to activated TCR/CD3 complexes.

IFN-gamma-stimulated enhancement of MHC class II antigen expression by the human mast cell line HMC-1.

The expression of MHC class II molecules by human mast cells has been reported in immunohistochemical surveys of inflammatory conditions, such as in tuberculin hypersensitivity. While these data suggest that human mast cells may act as antigen-presenting cells under inflammatory conditions, the induction of class II antigens on human mast cells has not been examined. In this study, we determined the effects of the inflammatory cytokines IFN-gamma and IL-4 on the expression of class II antigens HLA-DR, -DP, and -DQ by the human mast cell line HMC-1. HMC-1 cells were incubated with or without 1000 U/ml recombinant human IFN-gamma (rhIFN-gamma) and IL-4 (rhIL-4) for 72 hr and analyzed for expression of MHC class II antigens by direct immunofluorescence and flow cytometry. HMC-1 cells expressed significant levels of HLA-DR and moderate levels of HLA-DP and -DQ at baseline and when cultured without exogenous cytokines. Stimulation by rhIFN-gamma for 72 hr significantly increased the levels of HLA-DR and -DP expression but did not affect levels of HLA-DQ. Stimulation by rhIL-4 for 72 hr had minimal effect on expression of class II molecules, but induced a significant difference in levels of ICAM-1 (CD54) expression, indicating that this cytokine is involved instead in the control of certain accessory molecules. Our data showing constitutive expression of MHC class II molecules on HMC-1 cells and upregulation of that expression by rhIFN-gamma suggest that human mast cells function as antigen-presenting cells at sites where inflammatory cytokines are present.