Gold Coating of Respiratory Cilia for Scanning Electron Microscopy

The optimal thickness of gold coating of cilia for scanning electron microscopy was studied using respiratory mucosa obtained from pigs. We tested 8 different coating times, from 10 seconds to 4 minutes, which resulted in gold layer thicknesses varying from 16 ± 1 nm to 100 ± 3 nm. The thickness of the gold layer with a coating time of 60 seconds and voltage of 2.5 kV was 43 ± 5 nm. This thickness of gold layer gave good image quality without causing any electric charging. With thinner gold films, the amount of electric charging increased. When the coating time was longer, the gold layer was thicker but image quality did not improve. The thicknesses of the gold layers were measured using transmission electron microscopy (TEM)

Dew-worms in white nights: High-latitude light constrains earthworm (Lumbricus terrestris) behaviour at the soil surface

Soil is an effective barrier to light penetration that limits the direct influence of light on belowground organisms. Variation in aboveground light conditions, however, is important to soil-dwelling animals that are periodically active on the soil surface. A prime example is the earthworm Lumbricus terrestris L. (the dew-worm), an ecosystem engineer that emerges nocturnally on the soil surface. In the summer, the northernmost populations of L. terrestris are exposed to a time interval with no daily dark period. During a two-week period preceding the summer solstice, we studied the constraints that boreal night illumination imposes on L. terrestris surface activity by comparing their behaviour under ambient light with artificially-induced darkness. Looking for evidence of geographical divergence in light response, we compared the behaviour of native L. terrestris (Jokioinen, S–W Finland; 60°48′N) with two markedly more southern populations, from Preston (Lancashire, UK; 53°47′N) and Coshocton (Ohio, USA; 40°22′N) where the nights have a period of darkness throughout the year (total latitudinal range ca. 2300 km). Under ambient light conditions, L. terrestris emergence on the soil surface was diminished by half compared with the darkened treatment and it peaked at the darkest period of the night. Also mating rate decreased considerably under ambient light. The native dew-worms were generally the most active under ambient light. They emerged earlier in the evening and ceased their activity later in the morning than dew-worms from the two more southerly populations. The differences in behaviour were, however, significant mainly between native and UK dew-worms. In the darkened treatment, the behaviour of the three earthworm origins did not differ. Under the experimental conditions light condition was the dominant environmental factor controlling surface activity, but elevated night-time air temperature and humidity also encouraged dew-worm emergence without discernible differences among geographical origins. Our results show, that in boreal summer, the high level of night illumination strongly limits soil-surface activity of dew-worms. Considering the important regulatory role of L. terrestris in many ecosystem processes, this can have significant corollaries in dew-worm impacts on the environment. Although evidence for geographical differentiation in behaviour was obtained, the results point to phenotypic flexibility in L. terrestris light response

The role of outpatient visit after operative treatment of ankle fractures

It is a common practice that patients have a scheduled follow-up visit with radiographs following ankle fracture surgery. The aim of this study was to evaluate whether an early outpatient visit ( A change in treatment plan was observed in 9.8% of operatively treated ankle fracture patients. The mean age of the patients was 48 years and the mean follow-up time was 64 months. Of the changes in treatment plan, 91% were exclusively due to clinical findings such as infection. Only three of 878 patients required a change in their treatment plan based merely on the findings of the radiographs taken at the outpatient visit. Only 37% of the patients requiring a change in their postoperative management had solicited an unanticipated visit before the scheduled outpatient visit due to clinical problems such as infection or a cast-related issue. Our study showed that every tenth operatively treated ankle fracture patient requires a change in their treatment plan due to a clinical problem such as infection or a cast-related issue. Although at hospital discharge all patients are provided with written instructions on where to contact if problems related to the operated ankle emerge, only one third of the patients are aware of the clinically alarming symptoms and seek care when problems present. Our findings do not support obtaining routine radiographs at the early outpatient visit in an ankle fracture patient without clinical signs of a complication. (C) 2016 Published by Elsevier Ltd.Peer reviewe

The stroke oxygen pilot study: a randomized control trial of the effects of routine oxygen supplementation early after acute stroke--effect on key outcomes at six months

Introduction: Post-stroke hypoxia is common, and may adversely affect outcome. We have recently shown that oxygen supplementation may improve early neurological recovery. Here, we report the six-month outcomes of this pilot study. Methods: Patients with a clinical diagnosis of acute stroke were randomized within 24 h of admission to oxygen supplementation at 2 or 3 L/min for 72 h or to control treatment (room air). Outcomes (see below) were assessed by postal questionnaire at 6 months. Analysis was by intention-to-treat, and statistical significance was set at p#0.05. Results: Out of 301 patients randomized two refused/withdrew consent and 289 (148 in the oxygen and 141 in the control group) were included in the analysis: males 44%, 51%; mean (SD) age 73 (12), 71 (12); median (IQR) National Institutes of Health Stroke Scale score 6 (3, 10), 5 (3, 10) for the two groups respectively. At six months 22 (15%) patients in the oxygen group and 20 (14%) in the control group had died; mean survival in both groups was 162 days (p= 0.99). Median (IQR) scores for the primary outcome, the modified Rankin Scale, were 3 (1, 5) and 3 (1, 4) for the oxygen and control groups respectively. The covariate-adjusted odds ratio was 1.04 (95% CI 0.67, 1.60), indicating that the odds of a lower (i.e. better) score were non-significantly higher in the oxygen group (p= 0.86). The mean differences in the ability to perform basic (Barthel Index) and extended activities of daily living (NEADL), and quality of life (EuroQol) were also non-significant. Conclusions: None of the key outcomes differed at 6 months between the groups. Although not statistically significant and generally of small magnitude, the effects were predominantly in favour of the oxygen group; a larger trial, powered to show differences in longer-term functional outcomes, is now on-going. Trial Registration: Controlled-Trials.com ISRCTN12362720; Eudract.ema.europa.eu 2004-001866-4

Variations in pigment and carbohydrate content of gallbladder bile affect accurate quantitation of total protein when using the fluorescamine method

Background: Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients. Methods: Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method. Results: Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples. Conclusions: Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas

This article is available open access through the publisher’s website. Copyright @ 2009 The Authors.Background - Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. Methods - We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription–polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. Results - Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). Conclusions - We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.Sport Aiding Medical Research for Kids (SPARKS), Great Ormond Street Hospital/National Health Service, the National Cancer Institute and University of Parma