Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy

OBJECTIVES: To determine whether antibiotic prophylaxis reduces respiratory tract infections (RTI) and overall mortality in an unselected adults intensive care population. SEARCH STRATEGY: Systematic literature search in peer-reviewed journals indexed in MEDLINE, examination of relevant proceedings of scientific meetings and personal contact with trialists. SELECTION CRITERIA: All randomised clinical trials (RCTs), published and unpublished, comparing different forms of antibiotic prophylaxis used to reduce RTIs and mortality in unselected adult intensive care units (ICUs) populations. DATA COLLECTION AND ANALYSIS: Out of the 32 RCTs eligible for this review data have been extracted from published reports and then complemented with information provided by study investigators for 29 trials. Data were available only from published reports in the remaining three RCTs. For each trial the following information has been sought: a) method of randomisation; b) use of blinding techniques; c) number of randomised patients; d) number of patients with RTIs; e) number of deaths; f) number of patients excluded from the published analysis; g) number of RTIs and number of deaths among excluded patients. Pooled estimates of treatment effects across trials have been calculated after grouping RCTs in two main, mutually exclusive, categories: a) 15 trials testing the effect of a combination of a topical and a systemic antibiotic against no prophylactic treatment; b) 17 trials where the experimental treatment was a topical antimicrobial preparation. Crude proportions of RTIs and mortality were used to calculate the overall treatment effect. We also computed the number of ICU patients who need to be treated in order to prevent one infection and one death. MAIN RESULTS: Overall 32 RCTs including 5639 patients were identified. Pooled estimates of the 15 RCTs (including 3273 patients) testing the effect of the topical and systemic antibiotic combination indicate a strong significant reduction of both RTIs (OR = 0.36, 95% CI = 0.30-0.43) and total mortality (OR = 0.80, 95% CI = 0.68-0.93). Five and 23 patients need to be treated to prevent one infection and one death, respectively, using this treatment. When data on the effect of the combination based on topical antimicrobials were pooled from the 17 available trials (including 2366 patients) a marked reduction on RTIs (OR = 0.57, 95% CI = 0.46-0.69) also emerged but no corresponding effect on overall mortality (OR = 1.01; 95% CI = 0.84-1.22) was found. CONCLUSIONS: After 15 years of clinical research this meta-analysis of 32 RCTs shows that a regimen of antibiotic prophylaxis based on a combination of a systemic and topical antibiotic can reduce both RTIs and overall mortality in ICU patients in a way that is both statistically significant and humanly worthwhile. Over and above their personal opinions intensivists should take this evidence into account when defining their policies.     

Photo-patterning of sensor surfaces with biomolecular structures: characterisation using AFM and fluorescence microscopy

The miniaturisation of biosensors has resulted in the need to develop techniques for the high resolution patterning of different biological molecules onto surfaces. In this paper, we describe a procedure for the selective deposition of antibodies using biological self-assembly with photo-activation of a bound ligand, and we will detail methods which may subsequently be used to characterise the resultant biomolecular constructs.     

Vector alkaline phophatase substrate Blue III: one substrate for brightfield histochemistry and high-resolution fluorescence imaging by confocal laser scanning microscopy

OBJECTIVE: To compare the intraocular pressure (IOP)-reducing effect of the fixed combinations of timolol 0.5% and latanoprost 0.001% or 0.005% after 4 weeks' treatment. DESIGN: Following a 1-week run-in period on timolol 0.5% once daily, 139 patients were randomized to once-daily treatment with a fixed combination of timolol 0.5% and latanoprost 0.001% (comb. 10) or latanoprost 0.005% (comp. 50) or to the individual monotherapies. The IOP was measured at inclusion and at 8 a.m., noon and 4 p.m. on days 1, 7 and 28. RESULTS: Comb. 10, comb. 50, latanoprost and timolol reduced IOP by 3.7, 6.1, 4.9 and 2.1 mmHg, respectively, from a baseline mean diurnal IOP (+/- SEM) of 24.8 +/- 0.5, 24.1 +/- 0.4, 25.2 +/- 1.2 and 24.8 +/- 0.9 mmHg, respectively. The difference in IOP reduction was significant between comb. 50 and comb. 10 (P < 0.001), latanoprost (P = 0.046) and timolol (P < 0.001) in favor of comb. 50. There was also a significant difference between latanoprost and timolol (P = 0.007), in favor of latanoprost. All treatments were generally well tolerated. CONCLUSION: This study indicates that a fixed combination of latanoprost 0.005% and timolol 0.5% could be useful in the treatment of glaucoma.     

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.     

Organizational and individual factors affecting mental health and job satisfaction: A multilevel analysis of job control and personality.

This study evaluated variation at the individual and work unit levels in the relations of job control, hostility, and trait anxiety to mental health and job satisfaction. Questionnaire data from a sample of 2,900 employees working at 152 hospital wards were analyzed by means of multilevel regression analyses. Results showed that mental health (General Health Questionnaire-12), varying mainly at the individual level, was explained mostly by hostility and trait anxiety. Job satisfaction varied significantly at the individual and the ward level. Job control accounted for most of this multilevel variation. Thus, this study demonstrated the significance of individual characteristics and organizational effects in explaining the mental health and job satisfaction of employees. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Characterization of pigmented granules in minocycline-induced cutaneous pigmentation: observations using fluorescence microscopy and high-performance liquid chromatography

Ambiguous abdominal situs, asplenia/polysplenia and severe cardiac malformations characterize heterotaxy in humans. These anomalies result from the inability of the developing embryo to establish normal left-right asymmetry. We have studied an interesting family in which the heterotaxy phenotype segregates as an X-linked recessive trait. In order to map the heterotaxy locus (HTX), we have analysed 39 family members using highly-polymorphic microsatellite markers from the X chromosome. One of these markers, DXS994, shows no recombination with the disease locus in 20 informative meioses. Linkage analysis results in a maximum lod score of 6.37. Current genetic and physical mapping data assign the order of loci in Xq24-q27.1 as cen-DXS1001-(DXS994, HTX)-DXS984-tel. These results establish the first mapping assignment of situs abnormalities in humans.     

Evaluation of plasmid DNA for in vivo gene therapy: factors affecting the number of transfected fibers

Gene transfer by intramuscular injection of plasmid DNA has potential application in gene therapy. We examined factors affecting the number of expressing fibers, in contrast to total expression, following injection of plasmid DNA. Barium chloride proved effective in inducing muscle necrosis and regeneration in mice, and this increased the number of fibers expressing a reporter gene. Coinjection of ion-channel modulators did not increase the number of positive fibers, but increasing dose and repeated administration of plasmid did. Importantly, the plasmid size (7-16 kb) did not affect the number of fibers expressing the transgene, in both normal and regenerating muscle.     

Innocuous labeling of the subfragment-2 region of skeletal muscle heavy meromyosin with a fluorescent polyacrylamide nanobead and visualization of individual heavy meromyosin molecules

We have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.     

Genetic exchanges caused by ultraviolet photoproducts in phage lambda DNA molecules: the role of DNA replication

Personality development was examined in two groups of women studied since adolescence who were judged psychologically healthy at age fifty: 1) Independants, whose health improved from forty to fifty, were ambitious and intellectual. 2) Traditionals, healthy at both ages, were gregarious and nurturant. Traditionals showed steady personality growth since adolescence. Independents were constricted at age forty but recovered by fifty. These patterns are compared in terms of the fit between personality and sex role. Traditional personalitites fit conventional feminine roles, accounting for their health throughout the middle years. Independents improved when disengaging from mothering freed them to develop their more assertive skills.     

Primary carcinoid of the duodenum: detection and characterization by magnetic resonance imaging

We report a rare case of a primary small bowel carcinoid in which high-resolution MRI was able to detect a 1-cm primary duodenal neoplasm. This case illustrates that breath-hold contrast-enhanced MRI is capable of evaluating disease of the small bowel.     

The characterization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments using capillary electrophoresis

Mixtures of the polysaccharide derivatives, 19% hydroxyethylated SeaPrep agarose (SP-AG) and hydroxyethylcellulose (HEC), in aqueous buffer solutions are applied for the first time to the separation of DNA fragments using capillary electrophoresis (CE). These matrices form unique size-sieving networks that allow the separation of a wide size range of DNA fragments in a single analysis. Relative to their homogeneous counterparts, the composite separation matrices provide enhanced selectivity properties of DNA fragments, especially for fragments greater than 1000 base pairs (bp) in length. Additionally, the effects on separation performance of capillary temperature, the incorporation of a DNA intercalator, and applied field strength are demonstrated. Solution viscosity measurements of the homogeneous and composite matrix solutions were made in order to establish the entanglement threshold concentrations for the unique size-sieving solutions. The relatively low solution viscosities of the composite separation matrices allow reproducible replacement of the separation matrix between analyses. The mechanism of separation of DNA fragments for the composite matrices is proposed.     

Synthesis and characterization of YAG: Ce3+ fluorescence powders by co-precipitation method

YAG:Ce³⁺(Yttrium aluminum garnet) fluorescence powders were successfully prepared by co-precipitation method using aluminum nitrate, yttrium nitrate, cerous nitrate as the starting materials and ammonium carbonate as precipitant. The products were characterized by X-ray powder diffraction, luminescence spectrometer, transmission electron microscope (TEM). The XRD results showed that the obtained YAG:Ce³⁺ fluorescence powders had the crystalline structures of YAG at calcinations temperature of 900 °C and the TEM results showed that the grain diameters were about 100 nm. The YAG:Ce³⁺ fluorescence powders, synthesized by co-precipitation method, had the best luminescence property when the Ce doping amount was x=0.06 in the molecular formula of Y_3-xCe_xAl₅O₁₂, the calcinations time was 2 h and the calcinations temperature was 1000 °C.     

Racial and attitudinal factors affecting the perceptions of counselors by Black adolescents.

Presented to 247 Black male and female high school students racial and attitudinal information about a hypothetical male or female counselor and asked them to express their perceptions of the counselor. Attitudinal information about a counselor had a stronger effect than racial information on Ss' perception of the counselor: Counselors portrayed as attitudinally similar were rated significantly higher in attractiveness, trustworthiness, expertness, and social attraction than those portrayed as attitudinally dissimilar. Racial information also influenced perceived attractiveness: White counselors were rated higher than Black ones in attractiveness, although there was no difference in ratings of trustworthiness or expertise. White female counselors were perceived as more expert than their Black female counterparts, whereas the ratings of male counselors were not influenced by the racial variable. Implications for counselor–client relationships and the development of mental health services for minority populations are discussed. (33 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.     

Technology and older adults: factors affecting the adoption of automatic teller machines

Although technology can benefit service providers, caregivers, and the elderly, its application in an aging society can bring special challenges. This study looked at older adults' adoption of one technology that is highly prevalent in modern society--the automatic teller machine (ATM). The findings indicated that users and nonusers differed in mechanical reasoning skills and in attitudes toward ATM technology. Older adults with higher mechanical reasoning skills were more likely to be ATM users. Nonusers had more negative attitudes toward ATMs, and, among nonusers, those who had tried an ATM had more positive attitudes than those who had never tried one. The findings of this study are discussed in terms of factors that may affect the adoption of other technologies by older adults. Suggestions for increasing the acceptance of technologies by the elderly are also addressed.     

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.     

Oxidative modification of aldose reductase induced by copper ion. Factors and conditions affecting the process

Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(II)] through an oxygen-independent oxidative modification process. A stoichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inactivation. While metal chelators such as EDTA or o-phenantroline prevent but do not reverse the ALR2 inactivation, DTT allows the enzyme activity to be rescued by inducing the recovery of the native enzyme form. The inactive enzyme form is characterized by the presence of 2 equiv of bound copper, at least one of which present as Cu(I), and by the presence of two lesser equivalents, with respect to the native enzyme, of reduced thiol residues. Data are presented which indicate that the Cu-induced protein modification responsible for the inactivation of ALR2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of ALR2 and induces, through its oxidation to GSSG, the generation of an enzyme form linked to a glutathionyl residue by a disulfide bond.