Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Investigation of corneal autoantibodies in horses with immune mediated keratitis (IMMK).

Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional electrophoresis (2DE) with subsequent western blot analysis was performed followed by mass spectrometric identification of bands or spots of interest. Indirect immunofluorescence did not reveal a difference in immune response towards corneal proteins between healthy horses and those with IMMK. Using western blot analysis some horses affected by IMMK (4/28) showed a single band (1D) or a single spot (2DE) (5/28) not detected in healthy controls. The corresponding spot was identified as maspin (SERPINB5), a protein responsible for the inhibition of corneal vascularisation, cell migration and cell adhesion to the extracellular matrix. Tests with a recombinant human protein commercially available did not verify blot findings, but the human protein may not be fully cross-reactive. Still, maspin might play a role in some cases of equine IMMK. Further research is needed to clarify the etiology of this disease.

Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles.

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.

Sedation of children during measurement of CSF opening pressure: lack of standardisation in German children with pseudotumor cerebri.

BACKGROUND: Diagnosis of pseudotumor cerebri (PTC) requires proper documentation of raised CSF opening-pressure. In childhood results may not be reliable due to insufficient sedation/analgesia or drug effects. We aimed to evaluate the current practice regarding pain and stress management in children undergoing lumbar puncture (LP) for pressure measurement. METHODS: A one-year survey was conducted involving 368 German paediatric departments. All children with newly diagnosed PTC should be reported. Details analyzed here included: age, sex, CSF opening pressure and type of procedural sedation and analgesia (PSA) during LP. RESULTS: 61 patients were analyzed, aged 6 months to 17 years. 29 patients (47%) did not receive any kind of PSA. In children receiving PSA the following regimens were used: Ketamine; Midazolam; Ketamine + Midazolam; Midazolam + Piritramide; Propofol; Profofol + Midazolam; general anaesthesia. CONCLUSION: Pain and stress management in children undergoing LP for CSF opening pressure measurement is often insufficient. Pain, stress and the variability of PSA regimen may be confounders of pressure measurement. In order to prevent false diagnoses of PTC and to obtain comparable results at different centers, a general consensus on PSA in children undergoing LP for CSF opening pressure measurements is required.

[Evolution of breastfeeding pattern]. / Evolução do padrão de aleitamento materno.

OBJECTIVE: To follow the evolution of the breastfeeding practice among women in a childbirth clinic and evaluate the actions for its promotion. METHODS: Two cohorts of children born in an school hospital of Porto Alegre, Brazil, in different periods of time were compared regarding the prevalence of breastfeeding during the first six months of life, and the percentage of early cessation of breastfeeding. Both were prospective studies, including 202 children in the cohort of 1987, and 187 children in the cohort of 1994. All participants were healthy children, with birth weight of 2500g or more, were being breastfed and their parents were living in the same house. The 1987 study children were followed up through mail, and the 1994 study ones by home visits. RESULTS: Survival analysis showed similar frequencies of breastfeeding in the two cohorts. The prevalence of exclusive breastfeeding, although for a short period in both groups, was higher in the population studied in 1994, especially among children whose mothers were better educated. There was no rate difference of early cessation of breastfeeding between the two cohorts (36% for the 1987 cohort and 39% for the 1994 cohort). CONCLUSIONS: The results revealed a certain degree of apathy in regard of the promotion of breastfeeding during the studied period, giving support to actions to promote breastfeeding, especially among poor families.

Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins.

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

Optical methods for exploring dynamics of single copies of green fluorescent protein.

Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system.

Does parental breastfeeding knowledge increase breastfeeding rates?

OBJECTIVE: Although improving mothers' knowledge about breastfeeding can increase rates and duration of breastfeeding, little is known about the influence of fathers' knowledge. The purpose of this study was to assess the knowledge of mothers and fathers about breastfeeding before and after receiving postpartum advice and its relationship to the frequency of breastfeeding. METHODS: A clinical trial was performed with mothers and fathers of normal children born at the Hospital de Clínicas de Porto Alegre, Brazil, between July 1994 and March 1995. The study intervention consisted of postpartum advice supplied by means of a video film discussing basic topics of breastfeeding, an explanatory leaflet, and open discussion after viewing the video. The first 208 couples comprised the control group, the next 197 comprised experimental group 1, and the remaining 196 comprised experimental group 2. Immediately after delivery, mothers and fathers in the three groups answered a test on breastfeeding knowledge; they completed the same test at the end of the first month. All families received home visits at the end of the first, second, fourth, and sixth months, or until breastfeeding ceased. Logistic regression was used to evaluate the association between the mothers' and fathers' knowledge and frequency of breastfeeding. RESULTS: Postpartum advice increased the breastfeeding knowledge of mothers and fathers. The mothers with the highest level of knowledge had a 6.5 times higher chance of exclusively breastfeeding at the end of the third month, and 1.97 times higher chance of continuing breastfeeding to the end of the sixth month compared with other mothers. The fathers' knowledge also significantly influenced breastfeeding rates. The children whose fathers knew more had a 1.76 higher chance of being exclusively breastfed at the end of the first month, and 1.91 higher chance of receiving maternal milk at the end of the third month. CONCLUSION: A simple, inexpensive strategy can increase the level of breastfeeding knowledge of mothers and fathers and, consequently, have a positive impact on the frequency of breastfeeding.

Prospective randomized trial of two different immunoadsorbers in severe systemic lupus erythematosus.

Experiences with extracorporeal therapy in systemic lupus erythematosus have been published over the last 20 years and more. In addition to plasmapheresis, specific adsorption columns have been developed to deplete pathogenic antibodies and immune complexes in the plasma of patients with SLE. We conducted a prospective randomized trial to compare the efficacy of a disposable adsorption column, which contained a specific ligand, phenylalanine (IMPH-350|Pt, Diamed, Köln, Germany) with a regenerable Ig-adsorbing column containing sheep anti-human antibodies (Ig-Therasorb|Pt, Therasorb, München, Germany). Twenty SLE patients inadequately controlled with corticosteroids, anti-malarials, azathioprine or cyclosporine A, had strong evidence of disease in general, or an organ related SLAM score. They were randomized to receive either a perfusion of IMPH-350, or Ig-Therasorb, immunoadsorbed with 2.5 ml of plasma, three times each. The treatment regime was repeated after 4 weeks when the response was limited or non-existent. Response was defined as a reduction in the SLAM score of at least 30%. SLAM scores in the IMPH group decreased from 14.3+/-5.6 to 9.2+/-6.2 after 1 month and to 9.4+/-3.9 after 6 months; corresponding scores in the Ig-Therasorb group were 18.3+/-5. 5 to 11.2+/-7.6, decreasing to 9.2+/-2.9. After 1 month, 8/10 patients in both groups showed a response; after 6 months, 5/10 patients in the IMPH-350 group and 8/10 in the Ig-Therasorb group fulfilled the response criteria. Reduction of dsDNA antibodies directly after treatment was 50.8+/-6.6% in the IMPH-350 group and 61.0+/-8.0% in the IgTherasorb group. Results indicate that immunoadsorption is an additional option in the treatment of severe SLE. Choice of type of immunoadsorber and immunosuppressive treatment has to take into account the severity and chronicity of common disease activity and organ involvement, as well as economic aspects.

[A simple strategy increases mother's knowledge of breastfeeding and improves the breastfeeding rates] / Uma estratégia simples que aumenta os conhecimentos das mães em aleitamento materno e melhora as taxas de amamentação.

OBJECTIVE: To assess mother's knowledge of breastfeeding before and after guidance supplied during the postpartum period and its relationship to the prevalence of breastfeeding.METHODS: A clinical trial was performed with 405 mothers of normal children born at Hospital de Clínicas de Porto Alegre from July to December 1994. The intervention consisted of guidance given by means of a video film discussing basic topics on breastfeeding, an explanatory leaflet and open discussion after the video. The first 208 mothers constituted the Control Group and the remaining 197 the Experimental Group. All mothers answered a question form for identification purposes and a test on previous knowledge regarding breastfeeding in the maternity ward. The mothers in both groups were followed by means of home visits at the end of the first, second, fourth and sixth months, or until they stopped breastfeeding. At the end of the first month the mothers were submitted to the same test given right after delivery. Logistic regression was used to evaluate the association between the mothers knowledge of breastfeeding and the prevalence of breastfeeding.RESULTS: The mothers who received the intervention (Experimental Group) had a significantly higher score in the tests on knowledge of breastfeeding at the end of the first month as compared with the mothers in the Control Group (17.0 versus 14.7). The intervention increased by 1.7 mothers chances of achieving a score above the average. The mothers whose scores were above the average had a 8.2 higher chance of being breastfeeding exclusively at the end of the third month and twice as high of still being breastfeeding at the end of the sixth month.CONCLUSION: Simple strategies to increase mother's knowledge regarding breastfeeding can have a positive impact on breastfeeding rates.